GSM5918781: 938/88PosRetONC; Mus musculus; RNA-Seq
The purification of cell-specific, ribosomally loaded RNA from retina tissue from Tam-induced Aldh1l1-cre/ERT2+; NuTRAP+ mice was achieved following established protocol, with slight modifications23,31,32. For each sample, two retinae were pooled and homogenized in 1.0 mL 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; Thermofisher), and 1X cOmplete, EDTA-free Protease Inhibitor Cocktail (#11836170001; Millipore Sigma) with a glass dounce tissue grinder set (#D8938; 10 times with pestle A only). The homogenate was transferred to a 2 mL round-bottom tube and centrifuged at 10,000 x g for 10 minutes at 4°C. After centrifugation, 150 µL of the supernatant 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; Thermofisher) were washed three times in 1 mL ice-cold low-salt wash buffer (50mM Tris, pH 7.5; 12mM MgCl2; 100mM KCl; 1% NP-40; 100?g/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 (50 mM Tris, pH 7.5; 12 mM MgCl2; 300 mM KCl; 1% NP-40; 100 ?g/mL cycloheximide; 1 mM DTT). Following the last wash, 350 µL of Buffer RLT (Qiagen) supplemented with 3.5 µL 2-? 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 (Thermofisher 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 on 25 ng of total RNA for the preparation of strand-specific sequencing libraries from each TRAP-isolated RNA sample (input and positive fraction) and from conventionally isolated RNA samples from brain (tissue), according to manufacturer's instructions. 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, the addition of a single 'A' base, and finally ligation of adapters. 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).