Stereotaxic Surgical Implantation of Electrodes

Cell-type specific translated-mRNA purification (TRAP), was performed as previously described (Heiman et al., 2008 (link)) with a few modifications. Each sample consisted of a pool of 2–3 mice. BAC-TRAP transgenic mice (Drd2- and Drd1a-EGFP) were sacrificed by decapitation. The brain was quickly dissected out and placed in a cold buffer and was then transferred to an ice-cold mouse brain matrix to cut thick slices from which the nucleus accumbens (NAcc) and the dorsal striatum (DS) were punched out using ice-cold stainless-steel cannulas. Each sample was homogenized in 1 ml of lysis buffer (20 mM HEPES KOH (pH 7.4), 5 mM MgCl₂, 150 mM KCl, 0.5 mM dithiothreitol, 100 μg/ml CHX protease and RNAse inhibitors) with successively loose and tight glass-glass 2-ml Dounce homogenizers. Each homogenate was centrifuged at 2,000 × g, at 4°C, for 10 min. The supernatant was separated from cell debris and supplemented with NP-40 (EDM Biosciences) to a final concentration of 1% and DHPC (Avanti Polar lipids) to a final concentration of 30 mM. After mixing and incubating on ice for 5 min, the lysate was centrifuged for 10 min at 20,000 × g to separate the supernatant from the insolubilized material. A mixture of streptavidin-coated magnetic beads was incubated with biotinylated protein L and then with GFP antibody that was added to the supernatant and incubated ON at 4°C with gentle end-over rotation. After incubation, beads were collected with a magnetic rack and washed 5 times with high-salt washing buffer (20 mM HEPES-KOH (pH 7.4), 5 mM MgCl₂, 350 mM KCl, 1% NP-40) and immediately placed in “RTL plus” buffer (Qiagen). The mRNA was purified using the RNase micro KIT (Qiagen). RNA integrity was checked with the Bionalyzer (agilent 2100 Bioanalyzer, Agilent RNA 6000 nano kit). Five nanograms of mRNA from each sample were used for retro-transcription, performed with the Reverse Transcriptase III (Life Technologies) following the manufacturer’s instructions.

The technology was developed in the Heintz lab at Rockefeller University (Heiman et al., 2008 (link)). Briefly, PFC samples were homogenized in the appropriate buffer (containing NP-40, KCl, Tris, MgCl2, cycloheximide, protease inhibitor cocktail, RNAse inhibitors and DTT) and centrifuged. Supernatant (40 μl) was collected for total-RNA (INPUT), and the remaining supernatant (approximately 400 μl) was transferred to a separate tube and treated with anti-EGFP antibodies and Agarose Plus A/G beads (Santa Cruz_sc-2003*) overnight. After centrifugation at 1200 RPM for 1 min, the supernatant was removed and the beads, containing ribosome-bound RNA (TRAP samples), were washed 4 times with a high-salt buffer solution (containing NP-40, KCl, Tris, MgCl2, cycloheximide, and DTT) and eluted with QIAZOL (Qiagen # 79306). TRAP-RNA and total-RNA samples were extracted using the miRNeasy Mini Kit (Qiagen # 217004), according to manufacturer’s instructions.

BAC transgenic mice that express enhanced green fluorescent protein fused to the N-terminus of the large subunit ribosomal protein L10a under the control of dopamine D1a or D2 receptor promoter (Drd1-EGFP-L10a or Drd2-EGFP-L10a), generated as described^{26 (link)}, were maintained as heterozygotes on a C57Bl/6J background. Experiments with both lines were run in parallel. All the experiments were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by Rockefeller University’s Institutional Animal Care and Use Committee. For all experiments, male and female mice were 2-3 month-old, generated by in vitro fertilization (Transgenic and Reproductive Technology Center, Rockefeller University). Animals were housed on a 12-h light-dark cycle, in stable conditions of temperature, with food and water ad libitum. Four mice were pooled for each sample, males and females were mixed, and the sex ratio was counterbalanced between groups. All conditions were run in triplicates.

The supernatant was complemented with NP-40 (final concentration 1% vol/vol) and 1,2-diheptanoyl-sn-glycero-3-phosphocholine (final concentration 30 mM) and incubated for 5 min. It was then centrifuged at 20,000 × g for 10 min 4 °C and the pellet was discarded. Immunoprecipitation was performed according to previous protocols^{26 (link),83 (link)}. First, magnetic beads coated with anti-GFP antibody were prepared as follows: 300 µL of Streptavidin MyOne T1 Dynabeads (Invitrogen) per sample were washed in phosphate-buffered saline (PBS), incubated 35 min at room temperature (RT) with 120 µg of biotinylated protein L in PBS, washed 5 times with bovine serum albumin (BSA) 30 g.L⁻¹ in PBS, incubated 1 h at RT with 100 µg of monoclonal anti-GFP antibodies (50 µg clone 19F7 + 50 µg clone 19C8, Memorial Sloan-Kettering Monoclonal Antibody Facility, New York) in the homogenization buffer containing 1% (vol/vol) NP-40, washed 3 times and finally resuspended in 200 µL of homogenization buffer complemented with 1% (vol/vol) NP-40.
Magnetic beads coated with anti-GFP antibodies were added to the homogenates. After the addition of Superasin (final concentration 200 U.mL⁻¹, Life Technologies) and Rnasin (final concentration 400 U.mL⁻¹, Promega), the samples were incubated for 16 h at 4 °C under gentle end-over-end rotation. After 4 washes with homogenization buffer complemented with 1% (vol/vol) NP-40 and 200 mM KCl (total concentration KCl 350 mM), the RNA was eluted with RLT Plus buffer from the RNeasy Plus Micro kit (Qiagen) and 10 µL.mL⁻¹ β-mercaptoethanol (10-min incubation at RT and vortex). Then the RNA was purified according to the manufacturer’s instructions, with an on-column DNAse-I digestion step. The quantity of RNA was determined by fluorimetry using the Quant-iT Ribogreen, and its integrity was checked using the Bio-Analyzer Pico RNA kit before library preparation.
Ten nanograms of RNA were used for reverse transcription, performed with the Nugen Ovation RNAseq v2 kit. cDNAs were quantified by fluorometry, using the Quant-iT Picogreen reagent, and ultra-sonicated using a Covaris S2 sonicator with the following parameters: duty cycle 10%, intensity 5, 100 cycles/burst, 5 minutes. Two hundred nanograms of sonicated cDNA were then used for library construction using the Illumina TruSeq RNA sample prep kit, starting at the End-Repair step, and following the manufacturer’s instructions. The libraries were quantified with the Bio-Analyzer High-sensitivity DNA kit, multiplexed, and sequenced on an Illumina HiSeq 2500 instrument. We obtained more than 40 million 50 bp paired-end reads per sample.

Three-week-old Astrocyte-TRAP mice of both sexes were intraperitoneally injected with PTZ at 60 mg/kg (Sigma, 5 mg/mL stock solution in normal saline). Mice showing persistent convulsions at 8–10 min were subjected to rapid decapitation using decapicones (Braintree Scientific) for harvesting the brains. Control mice were injected with 110 μL normal saline (Sal) and decapitated similarly after 10 min. The harvested brains were snap-frozen in liquid nitrogen and stored at −80°C for TRAP. Six mice (three per treatment) were used.
TRAP was performed as described (Heiman et al., 2008 (link)) with a few modifications. Briefly, the brains were homogenized in ice in a buffer (20 mM HEPES pH 7.4, 150 mM KCl, 5 mM MgCl2, 0.5 mM dithiothreitol, 100 μg/mL cycloheximide (CHX), protease inhibitors, and RNase inhibitors). The lysates were cleared by centrifuging at 2000 × g for 10 min at 4°C and treated with DHPC (to 30 mM, Avanti) and NP-40 (to 1%, Ipgal-ca630, Sigma) for 5 min in ice. Lysates were further cleared by centrifuging at 20,000 × g for 15 min at 4°C. A 1/10^th volume of the cleared lysate was saved as the input control and used to generate RNAseq samples, and the rest was mixed with protein L-coated magnetic beads (Invitrogen), previously conjugated with a mix of two monoclonal anti-GFP antibodies (Doyle et al., 2008 (link)), and incubated with rotation for 4 h at 4°C. Beads were washed 5 times with a high-salt buffer (20 mM HEPES pH7.4, 350 mM KCl, 5 mM MgCl2, 1% NP-40, 0.5 mM dithiothreitol, and 100 μg/mL CHX) and finally resuspended in 200 ul normal-salt buffer (150 mM KCl, otherwise as above).
RNA was extracted from the input and TRAP samples using Trizol LS (Life Technologies) and a purification kit (Zymo Research) then quality-tested using RNA Pico Chips and BioAnalyzer 2100 (Agilent Technologies). All RIN values were >8.

The 2 or 14 d post-LPS/HI mouse brains were harvested in ice-cold dissection buffer following previously described procedures to purify the cell-type specific translating RNAs 35 (link). The rabbit anti-GFP antibodies, chromatin immunoprecipitation (ChIP) grade (#ab290, Abcam), were used for immunopurification. The purification specificity from CCR2-TRAP mice has been characterized 9 (link). The cDNA library was generated with a TruSeq Stranded mRNA Library Prep Kit (Illumina) with Agencourt AMPure XP beads for PCR cleanup. Samples were loaded onto a NextSeq 500 mid-output 150 cycle cartridge and sequenced on a NextSeq 500 (Illumina).

bacTRAP was performed on hypothalami of male and female POMC^Dre Drd2^Cre R26-lx-rx-EGFP-L10a mice or male and female POMC^Dre R26-rx-EGFP-L10a mice. Animals were decapitated randomly fed at 11 to 13 weeks of age, brains were quickly removed, and hypothalami were dissected using a stainless steel brain matrix (World Precision Instruments). Samples were snap-frozen in liquid nitrogen and stored at –80°C until further processing. For POMC^Dre Drd2^Cre R26-lx-rx-EGFP-L10a mice, hypothalami of 18–20 animals with balanced sex proportions were pooled per replicate for a total of 3 replicates. For POMC^Dre R26-rx-EGFP-L10a mice, hypothalami of 3 animals (2 males, 1 female) were pooled per replicate for a total of 3 replicates. The method for purifying translating ribosomes was performed as described by Heiman et al. (59 (link)) with minor modifications: in brief, 375 μL Protein A Dynabeads (Invitrogen, catalog 10001) per replicate were washed 3 times in wash buffer I (20 mM HEPES/pH 7.4, 5 mM MgCl₂, 150 mM KCl, 1% Nonidet P-40, 0.5 mM DTT, and 100 μg/mL cycloheximide) and subsequently resuspended in 275 μL wash buffer I. An anti-GFP antibody mixture (50 μg of Heintz Lab TRAP anti-GFP 19C8 antibody, catalog Htz-GFP-19C8; RRID: AB_2716737 and 50 μg of Heintz Lab TRAP anti-GFP 19F7 antibody, catalog Htz-GFP-19F7; RRID: AB_2716736) was incubated with the beads overnight at 4°C with slow end-over-end mixing. The following day beads were washed 3 times in wash buffer II (20 mM HEPES/pH 7.4, 5 mM MgCl₂, 150 mM KCl, 1% Nonidet P-40, 0.5 mM DTT, and 200 μg/mL cycloheximide) and subsequently resuspended in 200 μL wash buffer II. For lysis buffer preparation 1 tablet of cOmplete mini EDTA-free protease inhibitor cocktail (Sigma-Aldrich, catalog 11836170001) was dissolved in 10 mL 20 mM HEPES/pH 7.4, 5 mM MgCl₂, 150 mM KCl, 0.5 mM DTT, 40 U/mL RNasin, and 100 μg/mL cycloheximide. Pooled hypothalami were homogenized in 1 mL lysis buffer on a rotating glass/teflon potter homogenizer (Braun Biotech, Potter S) at 4°C twice at 250 rpm and 9 times at 750 rpm. Homogenates were centrifuged in low binding microfuge tubes (Applied Biosystems, catalog AM12450) at 2,000g and 4°C for 10 minutes. Supernatants were consequently mixed on ice with Nonidet P-40 (AppliChem, catalog A1694,0250) and 1,2-diheptanoyl-sn-glycero3-phosphocholine (Avanti Polar Lipids, catalog 850306P) at a final concentration of 1% (w/v) and 30 mM, respectively; incubated for 5 minutes; and centrifuged for 10 minutes at 17,000g and 4°C. A total of 30 μL of the supernatant was snap-frozen in liquid nitrogen until RNA extraction and served as hypothalamic input sample for each respective IP in the analysis of translational profiling. The remaining supernatant was mixed on ice with 200 μL anti-GFP antibody-coated beads and incubated for 1 hour at 4°C with slow end-over-end mixing. Sample-bead complexes (IPs) were collected via magnet and washed 4 times with wash buffer III (20 mM HEPES/pH 7.4, 5 mM MgCl₂, 350 mM KCl, 1% Nonidet P-40, 0.5 mM DTT, 100 μg/mL cycloheximide). RNA of hypothalamic input and IP samples was extracted using the RNeasy Micro Kit (QIAGEN, catalog 74004). In brief, samples were eluted off the beads by adding 350 μL RLT buffer and incubating for 5 minutes at room temperature. Subsequently manufacturer’s instructions were followed without alteration. RNA integrity was assessed using a 2100 Bioanalyzer (Agilent).

Affinity purifications of translating ribosomes were performed as published before^{46 ,89 (link)–92 (link)}. Twelve-week-old NCD- and HFD-fed IR^wt/wt L10a-EGFP^+/− mice and NCD-fed IR^fl/fl L10a-GFP^+/− mice were injected in the lateral ventricle with AAV-Dio2-Cre virus and killed 4 weeks post virus injection. Hypothalami were quickly extracted using an ice-cold stainless brain matrix (World Precision Instruments). Arcuate and median eminence regions were collected, frozen on dry ice and stored at −80 °C. Four to seven hypothalami were pooled per replicate. Further steps of the immunopurification of L10a-GFP-tagged ribosomes and subsequent extraction of RNA were performed exactly as previously described^{91 (link)}. RNA was eluted in 10 µl of nuclease-free water. RNA integrity was assessed using an Agilent 2100 bioanalyzer and RNA concentration was measured using a Qubit fluorometer (ThermoFisher Scientific).

All mice (C57BL/6J wild type [The Jackson Laboratory, Bar Harbor, USA], Thy1-Flag-Ppargc1a [Thy1-Ppargc1a] mice on a C57BL/6J genetic background provided by D. Lindholm [Mudò et al., 2012 (link)], Eno2^Cre+ mice [The Jackson Laboratory], Ppargc1a^flx/flx mice [The Jackson Laboratory], and Chat-L10a-eGFP mice [Heiman et al., 2008 (link)]) were kept under specific pathogen-free conditions in the central animal facility of the University Medical Center Hamburg-Eppendorf, Hamburg, Germany. Neuron-specific knockout mice were generated by crossing Ppargc1a^flx/flx with Eno2^Cre+ mice. Mice were grouped housed in a facility with 55–65% humidity at 24 ± 2°C with a 12 hr light/dark cycle and had free access to food and water. Sex- and age-matched adult animals (10–20 weeks of age) were used in all experiments. Wild-type mice were allocated to EAE or healthy group by cages. All procedures were carried out in accordance with the ARRIVE guidelines (Kilkenny et al., 2010 (link)).

The Cx3cr1^CreER: Rpl22^HA mice were anesthetized with sodium pentobarbital (80 mg/kg, i.p.) and the lumbar spinal cord and subcortical regions of the brain anterior to the pons (including amygdala, hippocampus, thalamus, basal ganglia and hypothalamus, but not the cerebral cortex, cerebellum or brainstem) were microdissected on ice and snap-frozen in dry-ice. RNA was isolated from the fresh frozen samples using the TRAP (translating ribosome affinity purification) approach^{40 (link)} based on a published protocol^{34 (link)} with some modifications. Briefly, the tissue was homogenized in polysome buffer and the supernatant from the lysate was incubated with 50 µl of anti-HA magnetic beads (Miltenyi, Auburn, CA, USA) for 2 h at 4 ˚C. Bead bound RNA was recovered by magnet, washed with high salt buffer three times and purified using RNeasy Micro Kit (Qiagen, Valencia, CA, USA).

To perform ribosome affinity purification on NPCs, timed-matings between Six2^TGC or Six2^TGC;Tsc1 and EGFP/Rpl10a mice were set, and embryos harvested at E14 or P0. All subsequent procedures were performed with solutions containing 100 µg/ml cycloheximide. Kidneys were isolated in ice-cold PBS and the capsule removed. Approximately 4–6 kidneys were placed in 1.5 ml Eppendorf tubes and digested with 250 µl of 0.25% Trypsin/0.2% Collagenase (Sigma-Aldrich) at 20 °C in a Thermomixer R shaking at 1400 rpm. After 10–15 min, the kidneys were removed, and the single-cell suspension was rinsed twice with 750 µl of ice-cold 0.3% BSA/PBS. NPCs were labeled using cell-surface primary antibodies (1:250 biotin-labeled anti-ITGA8, R&D Systems, Minneapolis, MN, Catalog #BAF4076; 1:50 PE-conjugated anti-PDGFRA, Invitrogen, Carlsbad, CA, USA; Catalog # 12-1401-81) while rocking on ice for 30–60 min. Cells were rinsed twice in 750 µl of ice-cold 0.3% BSA/PBS and subsequently stained with Streptavidin APC (1:50, Invitrogen; Catalog #SA1005) while rocking on ice for 30–60 min. After two washes in 750 µl of ice-cold 0.3% BSA/PBS, the NPCs were isolated by FACS on a SH800S cell sorter (Sony Biotechnology Inc., San Jose, CA, USA) selecting for GFP^HI (Six2 and EGFP/Rpl10a-expressing NPC), ITGA8+ (NPC with some stroma labeling, but not nephron epithelia, which remain EGFP/Rpl10a+), and PDGFRA- (Stroma is PDGFRA+) cells. To minimize cell stress during sorting, selection of an appropriately wide flow cytometer nozzle (100 µm), and precoating of collection tubes with collection buffer solution (ice-cold 0.3% BSA/PBS) were applied. Purification of polysome-bound RNA from kidneys was performed as previously described^{84 (link)}. Briefly, 250,000–300,000 NPCs were homogenized in ice-cold lysis buffer (20 mM HEPES, 5 mM MgCl2, 150 mM KCl, 0.5 mM DTT, 1% NP-40), 100 µg/ml cycloheximide, RNase inhibitors (40 U/ml RNasin (Promega, Madison, WI, USA), 1 U/µl Recombinant RNase inhibitor (Takara Biosciences, Mountain View, CA, USA)), and protease inhibitors (1 tablet per 10 ml lysis buffer, complete mini EDTA-free protease inhibitor cocktail, Sigma) by vigorous pipetting. The samples were centrifuged at 2000g for 10 min at 4 °C to remove large debris. Supernatants were extracted with 1% NP-40 and 30 mM 1,2-diheptanoyl-sn-glycero-3-phosphocholine (DHPC, Avanti Polar Lipids, Alabaster, AL, USA) on ice for 5 min, and centrifuged at 20,000g. The clarified supernatant was incubated in low-salt buffer (20 mM HEPES, 150 mM KCl, 5 mM MgCl₂, 0.5 mM DTT, 1% NP-40), RNAse inhibitors (40 U/ml RNasin (Promega) and 1 U/µl Recombinant RNase inhibitor (Takara Biosciences)), and 100 µg/ml cycloheximide containing streptavidin/protein L-coated Dynabeads (ThermoFisher, Waltham, MA, USA) bound with 17 µg of anti-GFP antibodies (AB_2716736, Catalog #HtzGFP-19F7 and AB_2716737, Catalog #HtzGFP-19C8, Memorial Sloan Kettering Centre, New York, NY, USA) overnight at 4 °C with gentle agitation. After 24 h, the beads were collected using magnets for 1 min on ice, washed four times in high-salt washing buffer (20 mM HEPES, 350 mM KCl, 5 mM MgCl2, 0.5 mM DTT, 1% NP-40, 40 U/ml RNasin (Promega) and 1 U/µl Recombinant RNase inhibitor (Takara Biosciences), and 100 µg/ml cycloheximide) and collected with magnets. RNA was eluted from the samples and purified using a Qiagen (Hilden, Germany) microRNeasy RNA kit per manufacturer’s instructions. RNA integrity was assessed using an RNA PicoChip (Agilent Bioanalyzer, Santa Clara, CA, USA) and the Clontech SMARTer library kit V2 (Takara Bio, USA) was used for cDNA library construction. Libraries were sequenced on an Illumina HiSeq-2500 using 75 bp paired-end sequencing.

The TRAP technique was performed using a modified version of a previous study on hypothalami of mice described under ‘Study design of bacTRAP (EGFPL10a) mice’^{47 (link)}. One day before TRAP, 375 µl of Dynal Protein G magnetic beads (Invitrogen) was washed three times (1 ml) and resuspended in 275 µl of IP wash buffer (20 mM HEPES (pH 7.4), 150 mM KCl, 5 mM MgCl₂ and 1% NP-40). Then, 50 µg of two monoclonal anti-GFP antibodies (HtzGFP-19C8 and HtzGFP-19F7) from the Memorial Sloan-Kettering Cancer Center was added to the magnetic beads and incubated with slow end-over-end mixing overnight at 4 °C.
On the day of TRAP, Dynal Protein G magnetic beads were washed three times (1 ml) and resuspended in 200 µl IP buffer (20 mM HEPES (pH 7.4), 150 mM KCl, 5 mM MgCl₂, 1% NP-40, 0.5 mM dithiothreitol and 100 mg ml⁻¹ cycloheximide) to remove unbound anti-GFP. Ice-cold polysome extraction buffer (20 mM HEPES (pH 7.4), 150 mM KCl, 5 mM MgCl₂, 0.5 mM dithiothreitol, 100 mg ml⁻¹ cycloheximide, protease inhibitors and 40 U ml⁻¹ recombinant RNasin Ribonuclease inhibitor) was added to the samples containing mouse hypothalami and homogenized with a motor-driven Teflon glass homogenizer. Homogenates were centrifuged at 4 °C for 10 min at, 2000g to pellet cell debris. Supernatant was transferred to a new microcentrifuge tube and NP-40 (Applichem) and 1,2-diheptanoyl-sn-glycero-3-phosphocholine (DHPC; Avanti Polar Lipids) were added to the supernatant at a final concentration of 1% and 30 mM, respectively. After incubation on ice for 5 min, the clarified lysate was centrifuged for 10 min at 13,000g to pellet insoluble material. Next, 30 µl of supernatant (input) was snap frozen in liquid nitrogen until RNA extraction as a comparison to the immunoprecipitated sample. Then, 200 µl of anti-GFP-coated Dynal protein G magnetic beads was added to the supernatant, and the mixture was incubated at 4 °C with end-over-end rotation for 1 h. Beads were subsequently collected on a MagnaRack (Invitrogen), washed four times with high-salt polysome wash buffer (20 mM HEPES (pH 7.4), 350 mM KCl, 5 mM MgCl₂, 1% NP-40, 0.5 mM dithiothreitol and 100 mg ml⁻¹ cycloheximide). Input and IP beads were resuspended and incubated in RLT buffer (RNAeasy micro kit, Qiagen) for 5 mins at RT. Supernatant was removed from the IP beads and RNA extraction was performed according to Qiagen’s protocol, including in-column DNase digestion. RNA was resuspended in 10 μl nuclease-free water. RNA quantity and quality of input and IP were determined with a Qubit Fluorometer (Invitrogen) and Agilent 2100 Bioanalyzer.

We used a total of 9 mice aged 8 weeks and 9 litters of mouse embryos aged E16.5 from the Foxj1:Cre × EGFP-L10a Bacterial Artificial Chromosome (BAC) transgenic lines (N = 3 samples of LVChP pooled from 3- to 8-week-old mice or 3 litters of E16.5 embryos). Brain tissue was immediately dissected and used for TRAP RNA purifications^{38 (link)}. ChP tissues were dissected in ice-cold dissection buffer (1 × HBSS; 2.5 mM HEPES-KOH; 35 mM glucose; 4 mM NaHCO₃; 100 μg/mL CHX) and homogenized in lysis buffer (20 mM HEPES-KOH; 5 mM MgCl₂; 150 mM KCl; 0.5 mM DTT; 1× protease inhibitors; 40 U/mL Rnasin; 20 U/mL Superasin) at 900 rpm for 12 strokes on ice with Teflon pestles in glass tubes (Kontes). Post-nuclear supernatant was prepared by centrifugation at 4 °C for 10 min at 2000 × g. Post-mitochondrial supernatant was prepared after incubation with additional 1% NP-40 and 30 mM DHPC, followed by centrifugation at 4 °C for 10 min at 20,000 × g. Immunoprecipitation was performed with magnetic streptavidin beads (MyOne T1 Dynabeads; Thermo Fisher #65601) conjugated to biontinylated anti-GFP antibodies (clones 19C8 and 19F7) for 16 h at 4 °C with gentle end-over-end rotation. Beads were collected on a magnet on ice, washed four times with 1000 μL 0.35 M KCl wash buffer (20 mM HEPES-KOH; 5 mM MgCl₂; 350 mM KCl; 1% NP-40; 0.5 mM DTT; 100 μg/mL CHX). RNA was eluted in Stratagene Absolutely RNA Nanoprep Kit (Agilent #400753) lysis buffer (with β-ME) and purified according to kit instructions. RNA quality was assessed using Bioanalyzer Pico Chips (Agilent, 5067-1513) and quantified using Quant-iT RiboGreen RNA assay kit (Thermo Fisher Scientific R11490). Libraries were prepared using Clonetech SMARTer Pico with ribodepletion and Illumina HiSeq to 50NT single end reads. Sequencing was performed at the MIT BioMicroCenter.

The TRAP methodology was adapted from the original protocol from ‘Cell-Type-Specific mRNA Purification by Translating Ribosome Affinity Purification (TRAP)’ (37 (link)).

To evaluate cellular Ppp1r12b vs. Ppp1r1b expression in the brain, single-cell RNA (scRNA) sequencing data from mouse TH+ neurons (GSE108020) [44] (link), mouse Drd1+ or Drd2+ cells (GSE112177) in dorsal striatum [45] (link) and human cortex neurons (GSE67835) [46] (link) were downloaded from GEO datasets website. Mouse Drd2+ cells translational profiling (GSE141463) used a BAC transgenic Translating Ribosome Affinity Purification (BacTRAP) strategy, allowing cell type-specific profiling of complex tissue [47] (link). Gene activity was normalized with GAPDH in each cell (cells without GAPDH counts were excluded).
Pathway analysis was carried out using MetaCore database, as previously described [48] , [49] (link), [50] (link) to identify biologically relevant pathways.

Three Wt and 4 Cyfip1^+/– male mice were injected with pAAV-FLEX-EGFPL10a and pENN.AAV.CaMKII 0.4.Cre.SV40 in CA1. The site and extent of expression (through about 2/3 of the dorsal hippocampus) were confirmed in pilot studies. The hippocampus was dissected, and the GFP expression was used to pull down ribosomes as described (Heiman et al., 2008 (link)). Associated mRNAs were used to generate cDNA. Sequencing and analysis were conducted by GENEWIZ.

Three weeks following injection of retrograde HSV-Cre into NAc, Cre-dependent L10-GFP-expressing mice (Rosa26^eGFP/L10a) were sacrificed and brains were immediately dissected into 1-mm coronal sections. Transduced tissue from ventral hippocampi (vHPC) of both wild-type and FosB^fl/fl mice was collected using 14-gauge biopsy punches guided by a fluorescent dissecting microscope (Leica) and stored at −80 °C until processing (n = 3/group, 3–4 mice pooled per n). Polyribosome-associated RNA was affinity purified as previously described^{51 (link),52 (link)}. Briefly, tissue was homogenized in ice-cold tissue-lysis buffer (20 mM HEPES [pH 7.4], 150 mM KCl, 10 mM MgCl₂, 0.5 mM dithiothreitol, 100 µg/ml cycloheximide, protease inhibitors, and recombinant RNase inhibitors) using a motor-driven Teflon glass homogenizer. Homogenates were centrifuged for 10 min at 2000 × g (4 °C), supernatant was supplemented with 1% NP-40 (AG Scientific, #P1505) and 30 mM DHPC (Avanti Polar Lipids, #850306P), and centrifuged again for 10 min at 20,000 × g (4 °C). Supernatant was collected and incubated with Streptavidin MyOne T1 Dynabeads (Invitrogen, #65601) that were coated with anti-GFP antibodies (Memorial Sloan-Kettering Monoclonal Antibody Facility; clone names: Htz-GFP-19F7 and Htz-GFP-19C8, 50 µg per antibody per sample) using recombinant biotinylated Protein L (Thermo Fisher Scientific, #29997) for 16–18 h on a rotator (4 °C) in low salt buffer (20 mM HEPES [pH 7.4], 350 mM KCl, 1% NP-40, 0.5 mM dithiothreitol, 100 µg/ml cycloheximide). Beads were isolated and washed with high salt buffer (20 mM HEPES [pH 7.4], 350 mM KCl, 1% NP-40, 0.5 mM dithiothreitol, 100 µg/ml cycloheximide) and RNA was purified using the RNeasy MicroKit (Qiagen, #74004). In order to increase yield, each RNA sample was initially passed through the Qiagen MinElute™ column three times. Following purification, RNA was quantified using a Qubit fluorometer (Invitrogen) and RNA quality was analyzed using a 4200 Agilent Tapestation (Agilent Technologies). cDNA libraries from 5 ng total RNA were prepared using the SMARTer® Stranded Total RNA-Seq Kit (Takara Bio USA, #635005), according to manufacturer’s instructions. cDNA libraries were pooled following Qubit measurement and TapeStation analysis, with a final concentration ~7 nM.

Affinity purification of EGFP-tagged polysomes was done 3 or 4 weeks after virus injections (Nectow et al., 2017 (link)). Briefly, mice were separated into three biological replicate groups of 4–6 mice per group and euthanized. Brains were removed, and the lateral septum was dissected on ice and pooled. Tissue was homogenized in buffer containing 10 mM HEPES-KOH (pH 7.4), 150 mM KCl, 5 mM MgCl₂, 0.5 mM DTT, 100 μg/mL cycloheximide, RNasin (Promega, Madison, WI) and SUPERase-In (Life Technologies, Waltham, MA) RNase inhibitors, and complete-EDTA-free protease inhibitors (Roche) and then cleared by two-step centrifugation to isolate polysome-containing cytoplasmic supernatant. Polysomes were immunoprecipitated using monoclonal anti-EGFP antibodies (clones 19C8 and 19F7; see Heiman et al., 2008 (link)) bound to biotinylated-Protein L (Pierce; Thermo Fisher Scientific)-coated streptavidin-conjugated magnetic beads (Life Technologies). A small amount of tissue RNA was saved before the immunoprecipitation (Input) and both input and immunoprecipitated RNA (IP) were then purified using RNAeasy Mini kit (QIAGEN). RNA quality was checked using an RNA PicoChip on a bioanalyzer. RIN values > 7 were used. Experiments were performed in triplicates for each group. cDNA was amplified using SMARTer Ultralow Input RNA for Illumina Sequencing Kit and sequenced on an Illumina HiSeq2500 platform.

For each independent biological replicate, hippocampi from 8 to 10 Fmr1-cTag^Camk2a-Cre and 8–10 Fmr1-cTag (Cre negative) male mice aged postnatal day 28 to 32 were pooled by genotype. Hippocampi were dissected into HBSS (Hanks’ Balanced Salt solution) containing 0.1 mg/mL cycloheximide and crosslinked three times on ice as described previously (Darnell et al., 2011 (link)). Crosslinked material was collected by centrifugation and resuspended in 1.2 mL lysis buffer (1X PBS, 1% Igepal, 0.5% deoxycholate and 0.1% SDS with protease inhibitor). Material was homogenized by mechanical homogenization and frozen at −80°C to ensure full cell lysis. To prepare for immunoprecipitation, lysates were thawed and subject to DNase and RNase treatment as described previously (Moore et al., 2014 (link)), using 54 µl of RQ1 DNase (Promega) and RNase A (Affymetrix, 20 U/ml) at a final dilution of 1:1,666,666. Following RNase and DNase treatment, the lysate was clarified by centrifugation at 2000 x g for 10 min, followed by an addition spin of the supernatant at 20,000 x g for 10 min. The resulting supernatant was pre-cleared by the addition of 200 µl protein G dynabeads (washed in lysis buffer). The sample was rotated for 45 min at 4°C. The resulting supernatant was used for immunoprecipitation with 200 µl of Protein G Dynabeads (Invitrogen) loaded with 25 µg each of mouse monoclonal anti-GFP antibodies 19F7 and 19C8 (Heiman et al., 2008 (link)). Immunoprecipitation was performed for 1.5–2 hr. After which the following washes were performed: twice with lysis buffer, twice with high salt lysis buffer (5X PBS, 1% Igepal, 0.5% deoxycholate and 0.1% SDS), twice with stringent wash buffer (15 mM Tris pH 7.5, 5 mM EDTA, 2.5 mM EGTA, 1% TritonX-100, 1% NaDOC, 0.1% SDS, 120 mM NaCl, 25 mM KCl), twice with high salt wash buffer (15 mM Tris pH 7.5, 5 mM EDTA, 2.5 mM EGTA, 1% TritonX-100, 1% NaDOC, 0.1% SDS, 1M NaCl), twice with low salt wash buffer (15 mM Tris pH 7.5, 5 mM EDTA), and twice with PNK wash buffer (50 mM Tris pH 7.4, 10 mM MgCl2, 0.5% NP-40). The second of each wash was rotated for 2–3 min at room temperature. RNA tags were dephosphorylated as described previously (Moore et al., 2014 (link)) and subjected to overnight 3’ ligation at 16°C with a pre-adenylated linker (preA-L32) (Moore et al., 2018 (link)) with the following ligation reaction: 2 µl of 25 µM linker, 2 µl of T4 RNA Ligase two truncated K227Q (NEB), 1X ligation buffer (supplied with ligase), 2 µl Superasin RNase inhibitor (Invitrogen), and 8 µl PEG8000 (supplied with ligase). The beads were washed and the RNA-protein complexes ³²P-labeled, and subjected to SDS-PAGE and transfer as described (Moore et al., 2014 (link)). Tags were collected from nitrocellulose as described (Zarnegar et al., 2016 (link)) with the following exceptions: Phenol:Chloroform:IAA, 25:24:1 pH 6.6 was used for extraction, and tags were precipitated with a standard sodium acetate precipitation. Cloning was performed using the BrdU-CLIP protocol as described (Moore et al., 2018 (link)) with a few exceptions. RT primers were generated with six nucleotide barcode index sequences to allow for up to 24 samples to be pooled together in one MiSeq run and subsequently demultiplexed (see Supplementary file 4 for sequences). To increase the yield for low-input samples, the Fmr1-cTag^Camk2a-Cre hippocampi samples were pooled (after reverse transcription) with parallel Fmr1-cTag^Camk2a-Cre cortex samples. Both Fmr1-cTag^Camk2a-Cre and Fmr1-cTag (Cre negative) samples were also included in the pool.

Gene expression in different brain cell types was analyzed by the translating ribosome affinity purification (TRAP) method as previously described [78 (link), 116 (link)]. Briefly, transgenic mice expressing EGFP-tagged ribosomal protein L10a (EGFP-L10a) in endothelial cells (Abcb1a-bacTRAP ES3026), pericytes (Gdnf-bacTRAP), astrocytes (Aldh1l1-bacTRAP JD130), oligodendrocytes (Olig2-bacTRAP JD97) L5b pyramidal cells (Colgalt2-bacTRAP DU9), or inhibitory interneurons (Dlx1-bacTRAP GM520) were used to affinity purify cell type specific polysomes from cortex. Each of these BAC transgenic lines has been described in detail previously and transgene expression can be viewed at www.gensat.org/TRAP_listing.jsp, except Gdnf-bacTRAP ES2243 which is described in detail in a forthcoming paper. Polysome-bound mRNAs from endothelial cells, pericytes, L5b pyramidal cells, interneurons, and mRNAs from whole tissue were analyzed by RNA sequencing. Oligodendrocyte TRAP mRNAs and matching whole tissue mRNAs were previously analyzed by microarray using the Affymetrix Mouse Genome 430 2.0 platform [78 (link)].

Exactly 90 min after the completion of behavioral training, rats (n = 10 per group) were deeply anesthetized with chloral hydrate (1.5 mg/kg) and perfused transcardially with 20 ml ice cold oxygenated artificial cerebrospinal fluid (ACSF) consisting of 125 mM NaCl, 3.3 mM KCl, 1.2 mM NaH₂PO₄, 25 mM NaHCO₃, 0.5 mM CaCl₂, 7 mM MgSO₄, and 15 mM glucose with 50 µM cycloheximide. Brains were quickly removed, blocked coronally around the amygdala and auditory cortex, and the two hemispheres separated and incubated in the perfusion solution for 4–5 min. Each hemisphere was then bisected along the rhinal fissure. The cortex of the dorsal half was peeled away from the underlying hippocampus and the area containing TE3 was dissected out. A smaller block containing the amygdala was dissected from the ventral half by peeling away the ventral hippocampus, trimming off the cortex lateral to the external capsule and trimming away the hypothalamus and medial portion of the striatum. The TE3 and amygdala blocks were quickly frozen in liquid nitrogen and stored at −80°C. Control and trained animals were run in parallel and tissue was collected in the middle of the animals’ light cycle.
The polysome purification and RNA extraction were performed according to published protocols (Heiman et al., 2008 (link); Kratz et al., 2014 (link)). TE3 or amygdala tissues from five animals were pooled (resulting in two biological replicates per group for sequencing), as pilot experiments found that this yielded sufficient mRNA. Samples were homogenized in 2 ml of ice-cold polysome extraction buffer [10 mM HEPES, 150 mM KCl, 5mMMgCl2, 0.5 mM DTT, one minitablet Complete-EDTA free Protease Inhibitor Cocktail (Roche), 100 µl RNasin Ribonuclease Inhibitor (Promega) and 100 µl SUPERase In RNase inhibitor (Ambion), 100 µg/ml cycloheximide] in douncer homogenizer. Homogenates were centrifuged for 10 min at 2000 x g at 4°C. The supernatants were clarified by adding 1% IGEPAL CA-630 (SigmaAldrich) and 30 mM DHPC (Avanti Polar Lipids) and incubated for 5 min on ice. The clarified lysates were centrifuged for 15 min at 20,000 x g at 4°C to pellet unsolubilized material, and 100 µl of the supernatant fluid was collected for isolation of the tissue transcriptome. The remainder was added to the conjugated beads/antibodies (200 µl) and incubated at 4C overnight with gentle agitation. The following day, the beads were collected with magnets for 1 min on ice, then washed in 1 mL 0,35M KCl washing buffer (20 mM HEPES, 350 mM KCl, 5mMMgCl₂, 0.5 mM DTT, 1% IGEPAL CA-630, 100 µl RNasin Ribonuclease Inhibitor and 100 µl SUPERase In RNase inhibitor, 100 µg/ml cycloheximide) and collected with magnets.
The conjugated beads/antibodies were freshly prepared before the homogenization on the day of the experiment by incubating 300 µl of Dynabeads MyOne Streptavidin T1 (ThermoFisher Scientific) with 120 µl of 1 µg/µl Biotinylated Protein L (ThermoFisher Scientific) for 35 min at room temperature with gentle rotation. Then, the conjugated protein L-beads were washed with 1XPBS and collected with magnets for three times. The conjugated protein L-beads were resuspended in 175 µl of 0.15M KCl IP wash buffer (20 mM HEPES, 150 mM KCl, 5mMMgCl₂, 0.5 mM DTT, 1% IGEPAL CA-630, 100 µl RNasin Ribonuclease Inhibitor and 100 µl SUPERase In RNase inhibitor, 100 µg/ml cycloheximide) and incubated for 1 hr at room temperature with 50 µg of each antibody. The beads were then washed 3 times with 0.15M KCl IP wash buffer and resuspended in the same buffer with 30 mM DHPC.
The RNA was extracted and purified with Stratagene Absolutely RNA Nanoprep Kit (Agilent Technologies, Santa Clara, CA) according to the manufacturer’s instructions. All the buffers were provided with the kit except otherwise specified. Briefly, the beads were resuspended in Lysis Buffer with ß-mercaptoethanol, incubated for 10 min at room temperature. 80% Sulfolane (Sigma) was added to the samples and the samples were mixed for 5–10 s, then added to an RNA-binding nano-spin cup and washed with a Low Salt Washing Buffer by centrifuge for 1 min at 12,000 x g at room temperature. DNA was digested by mixing the DNase Digestion Buffer and the samples for 15 min at 37C. Then, the samples were washed with High-Salt Washing Buffer, Low-Salt Washing Buffer and centrifuged for 1 min at 12,000 x g. Finally, the samples were eluted with Elution Buffer and centrifuge for 5 min at 12,000 x g at room temperature. The isolated RNA was stored at −80°C.

TRAP was conducted in homogenate samples of cortex obtained from AldH- EGFP-L10 control and stress mice, which express GFP in ribosomes of AldH+ cells, thus allowing for the immunoprecipitation of polysomes directly from astrocytes [20 (link)–22 (link)]. TRAP methods were conducted as previously published [27 (link)]. Twelve mice were used in total, six from each group. Briefly, mice were quickly decapitated and cortices were dissected on ice in a dissection buffer containing cyclohexamide (100 μg/ml, Sigma, dissolved in methanol, American Bioanalytical) in order to immediately freeze ribosome/mRNA (polysome) complexes. Samples were pooled (groups of two) and homogenized in extraction buffer [22 (link)] using a Teflon-glass homogenizer (Fisher #K8855100020) and then centrifuged for 10 min at 2000 × g at 4 °C. NP-40 (AG Scientific # P1505) and DHPC (Avanti # 850306 P) were added to the collected supernatant and incubated for 5 min on ice before centrifugation for 15 min at 20,000 × g until pelleted. The lysate supernatant was extracted, 30 mM DHPC was added, and then incubated with magnetic beads overnight. Magnetic beads (Streptavidin MyOne T1 Dynabeads; Invitrogen # 65601) were previously coated with Protein L (Fisher # PI-29997) for 35 min at room temperature (1 µg/µl in 1× PBS), collected on a magnetic rack (DynaMag-2; Invitrogen #123-21D), and washed 5 times in 1× PBS containing 3% protease-free bovine serum albumin (Jackson Immuno #001-000-162) and then subsequently incubated with 50 µg each of anti-GFP antibodies (HtzGFP_04 (clone19F7) and HtzGFP_02 (clone 19C8): Memorial Sloan-Kettering Monoclonal Antibody Facility) overnight at 4 °C and then washed again, prior to being incubated with the lysate. Following antibody incubation, beads were again collected on a magnetic rack, and unbound fragments were also collected and saved for RNA extraction step as total homogenate controls. Bound fragments were washed in a polysome buffer [22 (link)] and then resuspended in lysis buffer (Stratagene #400753) followed by RNA cleanup and extraction. All RNA samples were extracted using Stratagene’s Absolutely RNA Nanoprep Kit (Stratagene #400753), and unbound and bound samples were snap frozen and stored at −80 until use.
RNA samples were sent to the Yale K.E.C.K facility for sample quality control as per their standard protocols: Total RNA quality was determined by estimating the A260/A280 and A260/A230 ratios by nanodrop and RNA integrity was determined by running an Agilent Bioanalyzer gel (RNA integrity numbers ranged from 9.8 to 10). See further details of library preparation, sequencing, and TRAPseq data analyses in supplemental methods.

mRNA from translating polysomes was purified from mouse cerebella at P56 as previously described (Heiman et al., 2008 (link); Zhu et al., 2016 (link)). Heterozygous transgenic mice carrying EGFP-tagged ribosomal protein L10a (Tg(Neurod1-Egfp-L10a) and (Tg(Pcp2-Egfp-L10a) (Heiman et al., 2008 (link)) were used to purify mRNA from granule and Purkinje cells, respectively.

The PAPERCLIP procedure was performed as previously described (Hwang et al., 2016 (link)). Mouse monoclonal anti-GFP clones 19F7 and 19C8 (Heiman et al., 2008 (link)) were used for immunoprecipitation. Individual cTag-PAPERCLIP libraries were multiplexed and sequenced on MiSeq (Illumina, San Diego, CA) to obtain 75-nt single-end reads. cTag-PAPERCLIP was performed in four replicates on P56 granule cells, in three replicates on P56 Purkinje cells and in three replicates on developing granule cells (P0 and P21). Due to low amount of RNA obtained by cTag-PAPERCLIP, we pooled 2 mice per replicate for cTag-PAPERCLIP on P21 and P56 granule cells (Neurod1-Cre; Pabpc1^cTag and Atoh1-Cre; Pabpc1^cTag mice), 7 mice per replicate for cTag-PAPERCLIP on P56 Purkinje cells (Pcp2-Cre; Pabpc1^cTag mice) and 10 mice per replicate for cTag-PAPERCLIP on P0 granule cell precursors (Atoh1-Cre; Pabpc1^cTag mice).

To label ribosomal proteins, we chose several candidates whose N- or C- terminus is exposed to the surface of ribosomes based on the eukaryotic ribosome structure (Ben-Shem et al., 2011 (link)). We first generated multi-copy transgenic lines for rpl-1, rpl-4, rpl-29, rpl-30 and rps-18. Homologs of rpl-1 and rpl-4 were used for labeling ribosomes in mice (Court et al., 2008 (link); Heiman et al., 2008 (link)). However, the overexpression of these proteins in mechanosensory neurons caused abnormal axon and soma morphologies. We suspect that such defects could be due to overexpression. We then focused on rpl-30 and rps-18, null mutations in which caused larval arrest. Extrachromosomal arrays over-expressing N-terminally tagged rps-18 partially rescued rps-18(0) larval arrest, whereas C-terminally tagged rps-18 or rpl-30 did not. In addition, we used rpl-29 as a component of the ribosome large subunit; rpl-29 null mutants (tm3555) did not have any behavioral or morphological phenotypes.

Translated mRNA purification was performed as previously described (Doyle et al. 2008 (link); Heiman et al. 2008 (link); Thomas et al. 2012 (link); Heiman et al. 2014 (link)), with the following modifications: approximately 1000 heads per replicate were collected and homogenized in lysis buffer [20 mM HEPES, pH 7.4, 150 mM KCl, 5 mM MgCl₂, 0.5 mM DTT, 100 μg/ml Cyclohexamide, 100 U/ml RNaseOUT (Invitrogen), 1 × Complete Protease Inhibitor (Roche)]. The sample was centrifuged at 4° for 10 min at 2000 × g and the supernatant was collected. 1,2-diheptanoyl-sn-glycero-3-phosphocholine (DHPC; Avanti Polar Lipids, Inc.) and NP-40 were added to a final concentration of 30 mM and 1%, respectively, to the supernatant and incubated on ice for 5 min. The lysate was then centrifuged at 4° for 10 min at 20,000 × g; 40 μl of the supernatant was retained and placed directly into 200 μl of Lysis Solution [Ambion MicroPoly(A) Purist Kit] to be used to analyze the input RNA fraction. The remaining supernatant was added to Dynabeads Protein G magnetic beads (Novex) conjugated to mouse anti-GFP (Memorial Sloan-Kettering Monoclonal Antibody Facility19C8/19F7). The lysate-bead slurry was incubated for 30 min at 4° followed by washing in Wash Buffer (20 mM HEPES, pH 7.4, 5 mM MgCl₂, 350 mM KCl, 1% NP-40, 0.5 mM DTT, and 100 μg/ml Cyclohexamide) at 4°. The IP was resuspended in Lysis Solution [Ambion MicroPoly(A) Purist Kit] and mRNA was directly isolated following the manufacturer’s protocol.

For the pull-down of ribosome-associated RNA we were inspired by the TRAP (translating ribosome affinity purification) protocol developed in the laboratory of Dr. Nathaniel Heintz [26 (link), 27 (link)]. In short, this protocol is based on the pull-down of ribosomes using the large subunit protein Rpl10a, which is fused with GFP. We modified this protocol in order to pull down the endogenous Rpl10a protein as this would allow us to sample translation without altering the endogenous translational machinery. Immediately before the analysis, ES and ELA cells were trypsinized and resuspended in the appropriate growth medium supplemented with 100 μg/mL cycloheximide in order to stall ribosomes. After a 15 min incubation at 37 °C, cells were recovered and washed with ice-cold PBS plus 100 μg/mL cycloheximide to remove traces of serum and medium. Each sample (for both ELA and ES cells) included approximately 20 million cells and we conducted all experiments in duplicate. The samples were resuspended in 1 mL ice-cold lysis buffer (20 mM HEPES/KOH pH 7.3, 150 mM KCl, 10 mM MgCl₂, 1 % IGEPAL CA 630 (Sigma), 0.5 mM DTT, 100 μg/mL cycloheximide, 1 pill/10 mL complete MINI EDTA free protease inhibitor (Roche), 3 μL/mL RNAsin RNAse inhibitor (Promega), 3 μL/mL SUPERASEin RNAse inhibitor (Life Techologies)) and incubated on ice for 10 min before being triturated in a chilled dounce homogenizer with approximately 20 strokes. After lysis was completed, cellular debris (mostly formed by nuclei and fragments of cell membrane) was removed by centrifugation at 2000 RCF for 10 min. After centrifugation, 0.1 volumes of 300 mM 1,2-diheptanoyl-sn-glycero-3-phosphocholine (DHPC, Generon Ltd) were added to each sample to further solubilize ribosomes, followed by a 10 min incubation on ice and by a second centrifugation at 20,000 RCF for 10 min. The final clarified lysate was transferred to a new low-retention tube and 0.1 volumes were removed to be used as “input” samples for the measurements of total RNA levels. To immunoprecipitate ribosomes, 16 μg of anti-Rpl10a antibody (Sigma, catalogue number WH0004736M1) or an equivalent amount of anti-GFP antibody (control, Sigma catalogue number G1544) were added to each sample and incubated overnight with end-to-end agitation at 4 °C. The following day, 200 μL of 30 mg/mL Protein-G Dynabeads (Life Technologies) were washed in PBS plus 0.1 % Tween 20 (PBST), blocked in PBST plus 1 % bovine serum albumin (BSA) for 1 h, and added to the lysate/antibody mix. Beads were incubated overnight at 4 °C with end-to-end agitation. The following day the precipitated ribosomes were washed four times in 1 mL high-salt buffer (20 mM HEPES/KOH pH 7.3, 350 mM KCl, 10 mM MgCl₂, 1 % IGEPAL CA 630 (Sigma), 0.5 mM DTT, 100 μg/mL cycloheximide) and RNA was extracted using the RNEasy Micro kit (Qiagen) as per the supplier’s protocol. The results were quantified by spectrophotometry (Nanodrop) and capillary electrophoresis (Bioanalyzer RNA Nano kit; Additional file 1: Figure S2b). RNA (2 μg) was used to prepare each RNA-seq library. Purified RNA was ribosome-depleted using the RIBO-Zero Gold kit (Illumina), which uses magnetic beads conjugated to rRNA-specific oligonucleotides to remove ribosomal RNA. The final purified RNA was analyzed by capillary electrophoresis (Bioanalyzer Pico Kit) and retro-transcribed and cloned into a sequencing library using the SCRIPTSeq v2 library preparation kit (Illumina) according to the supplier’s protocol. Sequencing libraries were evaluated by Bioanalyzer (to verify the size distribution of fragments) and quantified by quantitative PCR using primers specific for Illumina adaptors (KAPA library quantification kit). All eight libraries (two replicates for ELA and ES cells, both IP and input) were mixed at a similar final concentration before cluster generation and sequencing on a HiSeq 2500 instrument.

Adult (8–12 weeks old) Epyc-Cre mice under ketamine/xylazine (100/10 mg/kg) anesthesia received single bilateral stereotaxic injections of 0.25 µl AAV-FLEX-EGFPL10a virus (3.75 × 10¹² genome copies/ml) into the mPFC (1.54 AP from bregma, 0.4 ML, –1.80 DV from dura). Animals were sacrificed in a controlled CO₂ chamber 3 weeks after surgery, brains were rapidly dissected in ice-cold Hanks balanced salt solution containing 2.5 mm HEPES-KOH (pH 7.4), 35 mm glucose, 4 mm NaHCO₃, and 100 µg/ml cycloheximide. The cortex was isolated from the rest of the brain, and each hemisphere was split along the coronal plane at the level of the genu of the corpus callosum (∼1.6 mm AP from bregma). The rostral portion was saved as the “PFC” and used for TRAP. Tissue from three mice (male and female) was pooled for each sample, and three biological replicates were collected. Polysome immunoprecipitations (IPs) were carried out as previously described (Schmidt et al., 2012 (link); Heiman et al., 2014 (link)). Briefly, the tissue was homogenized in extraction buffer containing 10 mm HEPES-KOH (pH 7.4), 150 mm KCl, 5 mm MgCl₂, 0.5 mm dithiothreitol, 100 µg/ml cycloheximide, RNasin (Promega, Madison, WI), and SUPERas-In (Invitrogen, San Diego, CA) RNase inhibitors, and complete EDTA-free protease inhibitors (Roche, Basel, Switzerland), and then cleared by centrifugation at 2000 × g. IGEPAL CA-630 (NP-40, Sigma-Aldrich) and diheptanoyl phosphatidylcholine (Avanti Polar Lipids, Alabaster, AL) were both added to the S2 supernatant for a final concentration of 1% each, followed by centrifugation at 20,000 × g. Polysomes were immunoprecipitated from the S20 supernatant using 100 µg monoclonal anti-EGFP antibodies (50 µg each of clones 19C8 and 19F7; Heiman et al., 2008 (link)) bound to biotinylated protein L (Pierce, Thermo Fisher, Waltham, MA) coated streptavidin-conjugated magnetic beads (Invitrogen), and washed in high salt buffer containing 10 mm HEPES-KOH (pH 7.4), 350 mm KCl, 5 mm MgCl₂, 1% IGEPAL CA-630, 0.5 mm dithiothreitol, 100 µg/ml cycloheximide, and RNasin RNase inhibitors (Promega). IPs were carried out overnight at 4°C. Bound RNA was purified using the Absolutely RNA Nanoprep kit (Agilent, Santa Clara, CA). RNA was also purified from the pre-IP supernatant to serve as whole-PFC “input” samples. RNA quantity was measured with a Nanodrop 1000 spectrophotometer, and quality was assayed on an Agilent 2100 Bioanalyzer. Only samples with RNA integrity values >7.0 were used for qRT-PCR analysis. cDNA was synthesized from 15 ng of IP or input total RNA using the Ovation qPCR System (NuGEN Technologies, Carlos, CA) following the manufacturer’s protocol. qRT-PCR was performed on an Applied Biosystems StepOnePlus Fast Real-Time PCR System using commercially available Taqman assays (Table 2) and following standard cycling conditions (50°C for 2 min, 95°C for 10 min, then 40 cycles of 95°C for 15 s and 60°C for 1 min). Ten nanograms of cDNA was used for each qRT-PCR reaction, and technical triplicates were run for each of the biological triplicates from TRAP IP and input samples. The mean C_T for technical replicates was used for quantification. Data were normalized to Gapdh by the comparative C_T (2^–ΔΔCT) method (Livak and Schmittgen, 2001 (link)). Data are presented as mean ± SEM of biological triplicates. Statistical significance was calculated between the normalized expression values (2^–ΔCT) from the IP and input biological replicates for each gene by Student’s t test in Microsoft Excel.

All polysome purifications and mRNA extractions were performed as described previously (Heiman et al., 2008 (link)). Three adult mice, balanced for gender and 8–10 weeks old, were pooled for each sample, and three biological replicates were collected for each bacTRAP line. Briefly, cortices were rapidly dissected in ice-cold HBSS containing 2.5 mM HEPES-KOH (pH 7.4), 35 mM glucose, 4 mM NaHCO₃, and 100 μg/ml cycloheximide, homogenized in extraction buffer 10 mM HEPES-KOH (pH 7.4), 150 mM KCl, 5 mM MgCl₂, 0.5 mM DTT, 100 μg/ml cycloheximide, RNAs in RNAse inhibitors (Promega, Madison, WI) and complete-EDTA-free protease inhibitors (Roche, Madison, WI), and centrifuged at 2000×g for 10 min. The supernatant from the homogenate was supplemented with a mix of detergents DHPC (Avanti Polar Lipids, Alabaster, AL) and NP-40 (Sigma–Aldrich, St Louis, MO), and cleared by a second centrifugation at 20,000×g for 15 min. Polysomes were immunoprecipitated with 100 μg of monoclonal anti-EGFP 19C8 and 19F7 antibodies bound to Protein-L conjugated Streptavidin MyOne Dynabeads overnight at 4°C and washed with high salt buffer containing 10 mM HEPES-KOH (pH 7.4), 350 mM KCl, 5 mM MgCl₂, 1% NP-40, 0.5 mM DTT, 100 μg/ml cycloheximide, and RNAs in RNAse inhibitors (Life Technologies, Grand Island, NY). Bound RNA was extracted and purified using Absolutely NanoPrep RNA purification kit (Stratagene, La Jolla, CA). RNA quantity was measured using Nanodrop 1000 spectrophotometer (Wilmington, DE) and quality determined with Agilent 2100 Bioanalyzer. RNA from 50 μl of homogenate prior to immunoprecipitation was also purified as Cortex input samples.

Brains were processed identically with MultiBrain Technology (NSA, NeuroScience Associates, Knoxville, TN) for DAB immunohistochemistry with a 1:75,000 dilution of Goat anti-EGFP serum (Heiman et al., 2008 (link)) according to the Vectastain elite protocol (Vector Labs, Burlingame, CA). Serial sections were digitized with a Zeiss Axiosko2 microscope at 10× magnification. For immunofluorescent staining, mice were deeply anesthetized using CO₂ chamber and transcardially perfused with 10 ml of phosphate-buffered saline (PBS) followed by 30 ml of 4% paraformaldehyde (PFA) in PBS. Brains were post-fixed in 4% PFA for 1 hr and cryoprotected by sequential sinking in 5% wt/vol sucrose in PBS at 4°C for 24 hr with gentle agitation followed by 30% wt/vol sucrose in PBS for the next 24 hr. 40-µm coronal sections were cut with Leica SM200R freezing microtome. Sections were blocked in 5% Normal Donkey Serum in PBS/0.1% Triton X-100 for 30 min and incubated overnight at 4°C with primary antibody against chicken anti-EGFP (Abcam, Cambridge, MA; 1:500), rabbit anti-WFS1 (Green Mountain Antibodies; 1:1000), mouse anti-NeuN (Millipore, Billerica, MA; 1:500), or anti-cFOS (Santa Cruz, Dallas, TX; 1:1000) in the blocking buffer. Appropriate Alexa dye-conjugated secondary antibodies were used at 1:400 dilution in the blocking buffer. Sections were mounted on the SuperFrost slides (VWR, South Plainfield, NJ) using Fluorogel media containing DAPI for nuclear counterstain (EMS, Hatfield, PA). All sections were imaged on Zeiss LSM-700 confocal microscope.

Collection of RNA was performed using a modified version of the TRAP protocol (Heiman et al., 2008 (link)), as described previously (Ainsley et al., 2014 (link); Drane et al., 2014 (link)). We have previously verified that this protocol results in low background when brain tissue is used from mice that do not express EGFP-L10a (Ainsley et al., 2014 (link); Drane et al., 2014 (link)). Briefly, a monoclonal anti-GFP antibody from the Monoclonal Antibody Core Facility at Memorial Sloan-Kettering Cancer Center (purified form of HtzGFP-19C8) was bound to magnetic epoxy beads (Invitrogen), and the beads were treated with bovine serum albumin to reduce nonspecific binding. For each mouse, three tissue punches were pooled, homogenized, centrifuged to pellet debris, and incubated with the beads at 4°C for 1 h. The supernatant (SN) was saved and the beads were washed five times with a KCl buffer. Trizol LS was used to extract RNA from the beads and the SN followed by back extraction to improve yield. Organic contaminants were removed with butanol and RNA was precipitated with isopropanol, NaOAc, and linear acrylamide overnight at −80°C. After two washes with 80% EtOH, RNA was resuspended in 10 ul of nuclease-free water.

RNA was isolated using a modified version of the TRAP technique (Doyle et al., 2008 (link); Heiman et al., 2008 (link)) that was optimized to remove background (Ainsley et al., 2014 (link)). Briefly, an anti-GFP antibody (HtzGFP-19C8) from the Monoclonal Antibody Core Facility at the Memorial Sloan-Kettering Cancer Center was covalently bound to magnetic epoxy beads (Invitrogen) followed by BSA treatment to reduce non-specific binding. Immediately following behavioral testing, Camk2a-TRAP mice were anesthetized using isoflurane. The hippocampi were rapidly dissected and rinsed in ice-cold dissection buffer (Heiman et al., 2008 (link)). Each hippocampus was added to 150 μl of homogenization buffer (Heiman et al., 2008 (link)) and homogenized using an automatic pestle. The tissue was centrifuged at 15,000 RPM for 10 min at 4°C. The lysate was transferred to a clean tube. For each sample, 1000 μg of protein was added to the prepared beads. Samples were incubated with the beads for 1 h at 4°C. The supernatant (SN) was saved for comparison to the immunoprecipitate (IP). After five washes with a KCl buffer (Heiman et al., 2008 (link)), RNA was extracted with Trizol LS. A back extraction was used to improve yield. Organic contaminants were removed with butanol and water-saturated diethyl ether washes (Krebs et al., 2009 (link)). RNA was precipitated using NaOAc, isopropanol, and linear acrylamide overnight at -80°C. After two washes with 80% EtOH, the RNA was resuspended in 15 μl nuclease-free water.

Ribosome captures were mainly carried out as already described (Heiman et al. 2008 (link)) with modifications to extract separate fractions enriched in cytosolic ribosomes or ER-bound ribosomes by centrifugation (Fig. 1A), before solubilization in NP40 (1%) and DHPC (diheptanoyl-sn-phosphatidylcholine, 30 mM) detergent and immunoprecipitation using magnetic beads coated with rabbit polyclonal anti-GFP antibody (Abcam, ab290). RNA was extracted using the PureLink RNA micro kit (Invitrogen). For details of the procedure and reagents, see Supplemental Methods.