The recombinant plasmids encoding the RNA scaffold and the aptamer sequences were linearized using the restriction enzymes referred to in Supplementary Figures S1 and S2 respectively. The in vitro RNA synthesis was carried out for 5 h at 37 °C using an Ampliscribe Kit (Epicentre, Madison, WI, USA). The residual plasmid DNA was degraded using 0.05 U/µL of DNase I (Epicentre) at 37 °C for 15 min. The RNAs were concentrated by ethanol precipitation and fractionated on 8% polyacrylamide gels containing 8 M urea (Thermo Scientific). The RNA bands were visualized by UV shadowing, using Fluor-coated TLC plates (Ambion Inc., Austin, TX, USA). The excised gel bands were frozen at −80 °C for 15 min, crushed using a pellet pestle (Thermo Scientific), and suspended in a TE buffer. Subsequently, the samples were heated at 73 °C for 15 min and a RiboGuardTM RNase inhibitor (Lucigen) (40 U/tube of in TE buffer) was added. The samples were incubated overnight at 37 °C with constant shaking. The extracted RNAs were recovered by filtration, using a 0.45 µm filter (Millipore, Billerica, MA, USA), followed by ethanol precipitation. The RNA pellets were resuspended in the TE buffer. By contrast, the RNAs were purified and concentrated using an RNA Clean & ConcentratorTM −5 kit (Zymo Research, Irvine, CA, USA).
Pellet pestle
Manufactured by Thermo Fisher Scientific
Sourced in United States, Switzerland, France
The Pellet Pestle is a laboratory tool used for homogenizing and grinding small samples, such as tissue or cell pellets, into a fine powder or paste. The pestle is designed to fit snugly inside a microcentrifuge tube or other small container, allowing for efficient and controlled sample preparation.
Automatically generated - may contain errors
Lab products found in correlation
Rhoa activation g lisa, by Cytoskeleton (2 mentions)
0.45 m filter, by Merck Group (1 mentions)
Dnase 1, by Illumina (1 mentions)
Rna clean concentratortm 5 kit, by Zymo Research (1 mentions)
G lisa, by Cytoskeleton (1 mentions)
Tri reagent, by Thermo Fisher Scientific (1 mentions)
Direct zol miniprep kit, by Zymo Research (1 mentions)
Immobilon fl transfer membrane, by Merck Group (1 mentions)
Irdye 800 goat anti rat igg, by LI COR (1 mentions)
Endo h and pngase f, by New England Biolabs (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
Centrifuge 5910 r, by Eppendorf (1 mentions)
Countess 2 fl, by Thermo Fisher Scientific (1 mentions)
Clariostar plus plate reader, by BMG Labtech (1 mentions)
Sds protease inhibitor cocktail type 1, by Roche (1 mentions)
Criterion system, by Bio-Rad (1 mentions)
Cell lytic mt mammalian tissue extraction reagent, by Merck Group (1 mentions)
Bradford protein assay, by Bio-Rad (1 mentions)
Chemiluminescence, by Cytiva (1 mentions)
Bca assay, by Thermo Fisher Scientific (1 mentions)
12 protocols using pellet pestle
1
In Vitro RNA Synthesis and Purification
2
RhoA Activation Assay for NAc Tissue
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Activated RhoA was determined with a RhoA activation G-LISA (Cytoskeleton # BK124, Denver, CO, USA) using materials provided in the kit. Three 14-gauge NAc tissue punches were homogenized with a Pellet Pestle (Thermo Fisher) in 100 μl lysis buffer. Homogenates were centrifuged (4°C, 13000 RPM, 1 min), and supernatants snap-frozen at −80°C for later processing. Lysates were thawed on ice, and protein concentrations were equalized with ice cold lysis buffer before following manufacturer’s instructions.
3
RhoA Activation Assay for NAc Tissue
4
RhoA Activation Assay Protocol
5
Isolation of High-Quality RNA from Orbital Muscle
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The orbital muscle samples were homogenized in TRI Reagent (ThermoFisher) using a handheld Pellet Pestle (Fisherbrand). After chloroform precipitation, RNA was isolated from the aqueous phase using a spin-column method (Direct-zol Miniprep kit, Zymo) according to the protocol which included an in-column DNAse step. RNA was quantified using Nanodrop 1000 software (v3.5.2, Inqaba Biotechnical industries) and RNA integrity (RIN) was evaluated using the PicoChip Bioanalyzer (Agilent 2100 Bioanalyzer). The muscle samples weighed 3–26 mg yielding 5–49 ng/µL RNA. Spectrophometric ratios suggested some impurities despite optimised RNA extraction protocols using phenol/chloroform precipitation, but were in the expected range for RNA isolated from muscle [16 (link)] with A260/280 range 1.6–1.9 and A260/230 range 0.4–1.6. RIN values ranged between 5.2 and 7.6.
6
Fly Head Protein Analysis by Western Blot
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For Western blotting, fly heads were homogenized in SDS sample buffer with a pellet pestle (Fisher, Pittsburgh, PA). Proteins were then fractionated by SDS–PAGE and transferred to Immobilon-FL transfer membranes (Millipore, Carrigtohill, Ireland) in Tris-glycine buffer. The blots were probed with primary antibodies against Rh1 (mouse, 1:2000; Developmental Studies Hybridoma Bank, Iowa City, IA) or INAD (rabbit, 1:2000, Wang et al., 2008 ), followed by IRDye 680 goat anti-mouse immunoglobulin G (IgG) or IRDye 800 goat anti-rat IgG (Li-Cor, Lincoln, NE) as secondary antibodies. Signals were detected using an Odyssey infrared imaging system (Li-Cor, Lincoln, NE).
All reagents for the Endo H and PNGase F digestions were obtained from New England Biolabs (NEB, Ipswich, MA). Twenty fly heads were collected and homogenized in glycoprotein denaturing buffer (1% SDS, 80 mM dithiothreitol). Homogenates were incubated for 4 h at 22°C and centrifuged to collect the supernatant. Digest reactions were processed according to manufacturer’s instructions with slight modification. Specifically, the reactions were incubated for 4 h at 37°C. All samples were solubilized in SDS loading buffer to equal volumes for Western blot analysis.
All reagents for the Endo H and PNGase F digestions were obtained from New England Biolabs (NEB, Ipswich, MA). Twenty fly heads were collected and homogenized in glycoprotein denaturing buffer (1% SDS, 80 mM dithiothreitol). Homogenates were incubated for 4 h at 22°C and centrifuged to collect the supernatant. Digest reactions were processed according to manufacturer’s instructions with slight modification. Specifically, the reactions were incubated for 4 h at 37°C. All samples were solubilized in SDS loading buffer to equal volumes for Western blot analysis.
7
Quantification of Symbiodiniaceae and Host Protein
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Each Aiptasia was homogenized with 500 µl filtered (0.22 µm) 2× phosphate buffered saline (PBS) in a 1.5 ml sterile Eppendorf tube using a cordless Pellet Pestle™ (Fisherbrand, USA). For the measurement of host protein content, 100 µl homogenate aliquots were centrifuged at 3000 × g for 3 min using Centrifuge 5910 R (Eppendorf, Germany). For each sample, three technical replicates of 5 µl of the five-fold diluted supernatant were transferred into a 96-well plate. The protein content was quantified using the Pierce™ Coomassie Plus (Bradford) Assay Kit (Thermo Scientific, US) following the manufacturer’s protocol and recording the absorbance at 595 nm using a CLARIOstar Plus plate reader (BMG LABTECH, Germany). For Symbiodiniaceae density, 100 µl aliquots of homogenate were centrifuged at 3000 × g for 3 min. 500 µl filtered 2× PBS was added to resuspend the Symbiodiniaceae pellets. The Symbiodiniaceae density was quantified for six technical replicate aliquots for each Aiptasia sample using a Countess II FL (Invitrogen, USA) fluorescence cell counter reading from channel CY5.
8
ENT1 Knockout Mouse Tissue Isolation
9
Protein Detection via Western Blotting
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Western Blotting for various proteins/targets was done via detection of chemiluminescence (Amersham Bioscience) as described previously (Takahashi et al., 2015) . Embryos or cultured cells were solubilized in TNE buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1 mM EDTA, 1% NP40, and 1 mM PMSF) for 10 minutes at 4°C, after performing 10-second pulse homogenization of pellet (Fisherbrand Pellet Pestle). Total protein concentration was determined using BCA assay (Thermo Fisher), and adjusted by addition of appropriate amount of SDS sample buffer to lysates. Lysates were subjected to SDS-PAGE electrophoresis and electro-transferred into PVDF (polyvinylidene difluoride) membranes. Bands corresponding to various proteins were detected by corresponding antibodies using ImageQuant LAS 4000 (GE Healthcare, Tokyo) and analyzed using ImageQuant software. Nuclear and cytoplasmic fractions were isolated using the NE-PER kit (Thermo Scientific 78833) according to the manufacturer's protocol.
10
Isolating Synaptosomal Fractions from BLA Tissue
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Synaptosomal fractions were obtained from BLA tissue using a previously established procedure (Bai & Witzmann, 2007) (link). Frozen brains were mounted on a cryostat and basolateral amygdala tissue was collected using a tissue puncher (Fine Science Tools). The tissue was homogenized using a Pellet Pestle (Fisher, #12141361) in 200 μL of homogenization buffer containing 20 mM HEPES, 1 mM EDTA, 2 mM EGTA, 320 mM sucrose, containing protease inhibitor (Roche, 05892791001) and phosphatase inhibitor (Roche, 04906837001) tablets. Homogenized tissue was centrifuged at 1000 g for 10 min. The supernatant was collected and centrifuged at 17,000 g for 15 min. The pellet was resuspended in 50 μL of homogenization buffer and layered on a sucrose gradient containing 100 μL of 0.8 M sucrose (1 mM EDTA, 2 mM EGTA) and 100 μL of 1.2 M sucrose (1 mM EDTA, 2 mM EGTA). This mixture was centrifuged at 54,000 g for 90 min. The layer between the 0.8 M and 1.2 M sucrose, containing the synaptosomal fraction, was collected and used for Western blotting following protein quantification with a BCA protein assay kit (Pierce).
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