title: GSM8527496: Replicate 5 Ipsi IP; Mus musculus; RNA-Seq
library_construction_protocol: Until reverse transcription steps, all work was completed with precautions against exogenous RNAses: equipment was cleaned with RNaseZap™ RNase Decontamination Solution (Invitrogen, AM9780) and rinsed with UltraPure™ water (Invitrogen, 10977015), all reagents were RNAse-free, and work completed on ice. Seven replicates of three pooled mice were processed bilaterally for each region of interest. Both unprecipitated input RNA and polysome immunoprecipitated (IP) samples were prepared for a total of 28 libraries. Two libraries were excluded due to non-Layer 5b and non-neuronal marker expression, presumably due to failed pulldowns. Ultimately, two replicates from male mice and three from female mice were used for the analysis. All 15 spinal cords were cleared with CUBIC tissue clearing and imaged coarsely; however, because the tissue was not perfused with PBS, the autofluorescence from the remaining blood made the injection site less clear than in perfused tissue. An unused mouse was cleared, and the cortex and spinal cord were imaged thoroughly to ensure that the injection protocol produced unilateral expression and the previously observed cortical distribution. Each mouse was rapidly decapitated, and the cortices ipsilateral and contralateral to the viral injection site were rapidly dissected in an ice-cold buffer consisting of 1X HBSS, 2.5 mM HEPES-KOH (pH = 7.4), 35 mM glucose (Sigma Aldrich, G7528-250G), 4 mM NaHCO3 (Sigma Aldrich, S6297-250G), and fresh 100 μg/ml cycloheximide (Sigma Aldrich, C7698-5G) in UltraPure™ water (Invitrogen, 10977015). Each tissue section was rewashed in the dissection buffer to remove excess blood. The triplicate hemicortices were then transferred to tissue homogenizers (DWK Life Sciences, K885510-0020) with 1 mL each of ice-cold homogenization buffer consisting of 10 mM HEPES-KOH (pH 7.4) (Thermo Fisher Scientific, AM9640G), 150 mM KCl (Thermo Fisher Scientific, AM9640G), 5 mM MgCl2 (Thermo Fisher Scientific, AM9530G), fresh 0.5 mM DTT (Sigma Aldrich, D9779-5G), 1 tablet per 10 mL  cOmplete™ EDTA-free Protease Inhibitor Cocktail (Roche, 11836170001), 400 Units/mL RNasin® Recombinant RNase Inhibitor (Promega, N2511), 200 Units/mL SUPERase•In™ RNase inhibitor (Invitrogen, AM2694), and 100 μg/ml cycloheximide (Sigma, C7698-5G) in UltraPure™ water (Invitrogen, 10977015). Tissues were homogenized with Teflon tissue homogenizing pestles (DWK Life Sciences, K886000-0019, 0.1–0.15mm clearance) using a lab stirrer (Yamato Scientific Co, 231349) for three strokes at 300 RPM. Then, the rotational speed was increased to 900 RPM for 12 additional strokes. The homogenate was transferred into pre-chilled 1.5 mL centrifuge tubes, spun at 2000 x g for 10 minutes at 4°C, and then transferred to a fresh 1.5 mL centrifuge tube. The homogenate volume was measured at this point, and 1/9th of this volume of 10% NP-40/IGEPAL was added (Sigma, I8896-50ML). Based on the resultant volume of this addition, 1/9th a volume of 300 mM DHPC (Avanti Polar Lipids, 850306P) was added. The samples were gently mixed by inversion five times and incubated on ice for 5 minutes. Then, the samples were spun at 20,000 x g for 15 minutes at 4°C. Ribosomal Pulldown: TRAP was performed as previously,87 albeit with a low-yield protocol consisting of half the standard amount of antibodies and beads in anticipation of the potential for differing pulldown efficiencies between neurons of differing abundance. The rationale was that by pooling mice and restricting bead-antibody conjugates, the beads might saturate equally between the differing amounts of available GFP and yield equivalent pulldowns. The magnetic beads were prepared during the previous day and in tandem with sample preparation. 150 μl for every sample of Streptavidin MyOne T1 Dynabeads (Invitrogen, 65601) were resuspended and washed 1X PBS. Each 500 μg vial of Pierce™ Biotinylated Protein L (Thermo Fisher Scientific, 29997) was resuspended on ice in 500 μl PBS. Each aliquot of beads was resuspended in 880 μl PBS and 120 μl Biotinylated Protein L and allowed to incubate at RT for 35 minutes with gentle end-to-end rotation. The beads were then washed five times with 1 mL 1X PBS containing 3% w/v nuclease-free BSA (Jackson Immunores. 001-000-162) and resuspended in 500 μl wash buffer containing 10 mM HEPES-KOH (pH = 7.4) (Fisher Scientific, AAJ16924AE), 150 mM KCl (Thermo Fisher, AM9640G), 5 mM MgCl2 (Thermo Fisher Scientific, AM9530G), 1% (v/v) IGEPAL® (Sigma Aldrich, I8896-50ML), 0.5 mM DTT (Sigma Aldrich, D9779-5G), 40 Units/mL RNasin® Recombinant RNase Inhibitor (Promega, N2511), 20 Units/mL SUPERase•In™ RNase inhibitor (Invitrogen, AM2694), and 100 μg/ml cycloheximide (Sigma Aldrich, C7698-5G) in UltraPure™ water (Invitrogen, 10977015). To this was added 50 μg total of mouse α-EGFP clones 19C8 and 19F7 mAb (25 μg each) (Memorial Sloan-Kettering Antibody and Bioresource Core Facility). The beads were then incubated with slow tilt rotation for >1 hr at RT. During the high-speed spin of the samples, the beads were washed thrice with 1 mL of the aforementioned 0.15 M KCl wash buffer and resuspended in 180 μl. 20 μl 300 mM DHPC (Avanti Polar Lipids, 850306P) was added to the resuspension. After the high-speed spin, 30 μl of supernatant was removed as “input” samples mixed with 350 μl buffer containing 340 μl RLT and 10 μl βME from the RNeasy Plus Micro kit (Qiagen, 74034) and flash frozen for parallel purification with other samples. The remaining supernatant was placed in a chilled 1.5 mL tube, mixed with 200 μl bead and antibody solution, and incubated on gentle rotation at 4°C overnight. The following morning, beads were magnetically collected on ice. 30 μl of supernatant was reserved as “unbound” RNA but ultimately not sequenced. The remaining supernatant was removed and washed four times with 1 mL of wash buffer containing 10 mM HEPES-KOH (pH = 7.4) (Fisher Scientific, AAJ16924AE), 350 mM KCl (Thermo Fisher Scientific, AM9640G), 5 mM MgCl2 (Thermo Fisher Scienific, AM9530G), 1% (v/v) IGEPAL® (Sigma Aldrich, I8896-50ML), 0.5 mM DTT (Sigma Aldrich, D9779-5G), 40 Units/mL RNasin® Recombinant RNase Inhibitor (Promega, N2511), and 100 μg/ml cycloheximide in UltraPure™ water (Invitrogen, 10977015). All samples, bound and unbound, were then suspended in 350 μl RLT-βME buffer as described above and incubated at room temperature on gentle end-over-end rotation for 10 minutes. The beads were magnetically separated, and the supernatant was removed and placed in fresh 1.5 mL tubes. RNA was purified with the RNeasy Plus Micro kit per the manufacturer's instructions but eluted into UltraPure™ water (Invitrogen, 10977015) rather than the provided elution buffer (Qiagen, 74034). RNA was quantified, and RNA integrity was confirmed with a Nanodrop spectrophotometer as well as a TapeStation High-Sensitivity RNA ScreenTape (Agilent, 5067-5579). Libraries were prepared using the Revelo™ RNA-Seq High Sensitivity library preparation kit (Tecan, 30201358) and associated SPIABoost™ Mouse reagents (Tecan, 30201371) and sequencing adapters (Tecan, S02317-FG) per manufacturer's instructions. Library fragment distribution was analyzed with a TapeStation D1000 DNA ScreenTape (Agilent, 5067-5582). Libraries were sequenced on a NextSeq 500 (Illumina) on a high-output paired-end 75 base pair run (Illumina, 20024907). A median of 35,208,430 paired-end reads passed the filter, ranging from 5,524,727 to 77,421,692 reads per sample.