GSM7845456: 2126_2137_Input; Mus musculus; RNA-Seq
Eyecup harvesting was performed within the 9:00-11:00 am time window for all experimental groups. The purification of cell-specific, ribosomally loaded RNA from eyecup tissue from Tam-induced Aldh1l1-cre/ERT2+; NuTRAP+ mice was achieved following an established protocol, with slight modifications40,42,50. Individual eyecups were placed in 1.5 ml tubes containing 150 µl ice-cold homogenization buffer (50 mM Tris, pH 7.4; 12 mM MgCl2; 100 mM KCl; 1% NP-40; 1 mg/ml sodium heparin; 1 mM DTT) supplemented with 100 µg/ml cycloheximide (#C4859-1ML, Millipore Sigma), 0.5 mM spermidine (#05292-1ML-F, Millipore Sigma), 200 units/ml RNaseOUT Recombinant Ribonuclease Inhibitor (#10777019; Thermo Fisher), and 1X cOmplete, EDTA-free Protease Inhibitor Cocktail (#11836170001; Millipore Sigma). Each eyecup was homogenized using plastic pestle and cordless motor (two pulses of 5 sec duration/each). After pooling homogenates from 4 eyecups (4 eyecups/2 mice = biological replicate), each sample was taken up to 1ml final volume with homogenization buffer and gently resuspended. After centrifugation (10,000 x g, 10 min, at 4° C), 100 µl of the supernatant of each sample was saved as input. The remaining supernatant was transferred to a 2 ml round-bottom tube and incubated with 5 µg/µl of anti-GFP antibody (ab290; Abcam) at 4°C with end-over-end rotation overnight. Dynabeads Protein G for Immunoprecipitation (#10003D; Thermo Fisher) were washed three times in 1 ml ice-cold low-salt wash buffer (50 mM Tris, pH 7.5; 12 mM MgCl2; 100 mM KCl; 1% NP-40; 100 μg/ml cycloheximide; 1mM DTT). After removal of the last wash, the homogenate/antibody mixture was transferred to the 2 ml round-bottom tube containing the washed Protein-G Dynabeads and incubated at 4°C overnight with end-over-end rotation. Magnetic beads were collected using a DynaMag-2 magnet. Beads were then washed three times with 1 ml of high-salt wash buffer (50mM Tris, pH 7.5; 12mM MgCl2; 300mM KCl; 1% NP-40; 100μg/ml cycloheximide; 1mM DTT). Following the last wash, 350 µl of Buffer RLT (Qiagen) supplemented with 3.5 µl 2-β mercaptoethanol was added directly to the beads and incubated with mixing on a ThermoMixer (Eppendorf) for 10 minutes at room temperature. The beads were magnetically separated and the supernatant containing the target bead-bound ribosomes and associated RNA was transferred to a new tube. 350 µl of 100% ethanol was added to the tube (“positive” fraction: enriched in translatome associated to EGFP-tagged ribosomes) and then loaded onto a RNeasy MinElute column. RNA was isolated using RNeasy Mini Kit (#74104, Qiagen), according to manufacturer's instructions. RNA was quantified with a Nanodrop One spectrophotometer (Thermo Fisher Scientific) and its quality assessed by HSRNA ScreenTape with a 4150 TapeStation analyzer (Agilent Technologies, Santa Clara, CA). The NEBNext Ultra II Directional Library Prep Kit for Illumina (#NEBE7760L; New England Biolabs Inc., Ipswich, MA) was used with 25 ng of total RNA for the preparation of strand-specific sequencing libraries from TRAP-isolated RNA samples (input and positive fraction) according to manufacturer's instructions40. Briefly, polyA containing mRNA was purified using oligo-dT attached magnetic beads. mRNA was chemically fragmented and cDNA synthesized. For strand-specificity, the incorporation of dUTP instead of dTTP in the second strand cDNA synthesis does not allow amplification past this dUTP with the polymerase. Following cDNA synthesis, each product underwent end repair process, A tailing and adapter ligation. The cDNA products were further purified and enriched using PCR to make the final library for sequencing. Library sizing was performed with HS RNA ScreenTape (#5067-5579; Agilent Technologies). The libraries for each sample were pooled at 4 nM concentration and sequenced using an Illumina NovaSeq S4 system (PE150) at the OMRF Clinical Genomics Facility.