Am2616

Mouse Brain Dissociation was performed as described by 10x genomics in the tissue fixation and dissociation for Chromium Fixed RNA Profiling. Briefly, fresh frozen mouse cerebellum was weighed and fixed in 4% paraformaldehyde solution for 2 hours at 2mL per 25mg of tissue with periodic agitation. Then, the tissue was centrifuged and re-suspended in 1x PBS twice. Washed tissue was resuspended in ice-cold 70% ethanol at 2mL per 25mg of tissue and incubated overnight at 4C. After incubation, the tissue was centrifuged and resuspended with 1x PBS twice. Tissue was then resuspended in 2mL dissociation buffer (160uL LiberaseTL enzyme - Millipore Sigma #5401020001 + RPMI) using the gentleMACS OctoDissociator with heaters (Miltenyi Biotec # 130-096-427) for 30 minutes at 50rpm. Nuclei were washed with 1x PBS + 0.02% BSA (Thermofisher #AM2616) + 0.2u/uL RNAse inhibitor and stained 1ul/ml DAPI(Thermofisher #62248) for 10 minutes. To remove excess debris, DAPI⁺ nuclei singlets were sorted using FACS before processing by HCRFlowFISH.
Nuclei were either stored for future use according to 10x Genomics Recommendation or proceeded directly into HCRFlowFISH.

In 20 μl, 3 μg recombinant His6-MBP or MBP-eIF2A-His6 was mixed with 20% RRL in 100 mM KCl, 10 nM amino acid mix minus methionine, 10 nM amino acid mix minus leucine, and 0.5 mM MgOAc (same as in vitro translation above) or with 0.27 μM purified 40S ribosomal subunits in 40S Binding Buffer (14 (link)) (final of 50 mM HEPES, 100 mM KCl, 5 mM MgCl₂,; pH 7.5) and were incubated at 25°C for 10 min. Indicated samples were then crosslinked with 0.25% (v/v) formaldehyde (Sigma # F79-500) for 30 min at 25°C and quenched with 20 μl 100 mM Tris–HCl, pH 7.5. Samples were diluted 1:10 in ice-cold Wash Buffer (20 mM Tris–HCl, 140 mM KCl, 10 mM MgCl₂, 0.1% (v/v) Triton X-100, 10 mM imidazole; pH 7.5) and added to 20 μl HisPur Ni-NTA Magnetic Beads (Thermo Fisher # 888831) for 30 min at 4°C with end-over-end rotation. For 40S ribosomal subunit and 80S ribosome binding experiments, HisPur Ni-NTA Magnetic Beads were blocked with 1 μg/μl BSA (Invitrogen # AM2616) and 2 μg/ml yeast tRNA (Thermo Fisher # AM7119) in Wash Buffer for 1 h at 4°C with end-over-end rotation. Beads were then washed 5× with 400 μl ice-cold Wash Buffer. Bound proteins were then eluted with 200 μl 2× reducing LDS sample buffer (BioRad # 1610747) and heating for 15 min at 70°C. 20 μl of eluate was analyzed by SDS-PAGE and Western blotting as described above.

Cells were collected 48 h after doxycycline induction, and washes were done in 1 × PBS with 0.04 mg ml–1 BSA (Thermo Fisher Scientific, AM2616). Clumps were removed by using a 30 μM CellTrics filter (cat. no. 04-004-2326). 25% iA (Tubb3:GFP) and 75% iALF or 25% iA (Tubb3:GFP) and 75% iALFiP were pooled as to separate libraries having 1,000 cells per μl. 10X Genomics Chromium Single Cell 3’ library kit was used to generate a single-cell library for a targeted cell recovery rate of 10,000 cells (Chromium i7 Multiplex Kit, Chromium Single Cell B Chip Kit v3, Chromium Single Cell 3’ GEM, Library and Gel Bead Kit v3). The libraries were sequenced on an Illumina Next-Seq 500 High Output using V2.5 chemistry with 26 × 98 bp – 150 cycles run confirmation at NYU Genomics Core facility.

Cells were fixed with 4% paraformaldehyde at room temperature for 10 min and then washed twice with PBS. 70% (v/v) EtOH was then added and cells were stored at 4°C overnight. Cells were then washed twice with wash buffer (2x SSC with 10% formamide in RNase-free water). Following aspiration of the wash buffer, cells were incubated with hybridization buffer (2x SSC, 10% v/v deionized formamide, 10% (w/v) dextran sulfate, 2 mM vanadyl ribonucleoside complex, 1 mg/ml yeast tRNA (Ambion AM7119), 0.005% BSA (Ambion AM2616) with 1 ng/μl 5′ labeled FAM-oligo(dT20) probes (Genelink 26-4620-02) at 37°C overnight. Cells were then washed 3 times with pre-warmed wash buffer at 37°C.