GSM5287433: WT_4_rep5; Mus musculus; RIP-Seq
Immunoprecipitation and purification of ribosome-associated RNA from pyramidal neurons was performed on Shank3(dC);HA-Rpl22(f/f);Camk2a-Cre(+/-) and Shank3(+/+);HA-Rpl22(f/f);Camk2a-Cre(+/-) mouse brains as described(23) with minor modifications. Briefly, mouse corti were homogenized in lysis buffer (50 mM Tris, 100 mM KCl, 12 mM MgCl2, 1% NP-40, 100 μg/ml cycloheximide, 10 mM Ribonucleoside Vanadyl Complex, 1 mM DTT, plus RNAsin and CompleteTM EDTA-free protease inhibitor mixture, pH 7.4) on ice.  Homogenates were spun at 10, 000 g for 10 min to remove debris. After collecting the supernatants, ribosomes and associated RNA were immunoprecipitated with an anti-HA antibody. Ribosome-associated RNA was purified by RNeasy mini kit (Qiagen), RNA-seq libraries were prepared using Illumina TruSeq stranded mRNA library preparation kit (Illumina). Library quality was validated by high sensitivity DNA analysis kit using Agilent Bioanalyzer 2100, and the concentration of RNA-seq libraries was quantified by qPCR using the KAPA library quantification kit (Kapabiosystems). Sequencing of RNA-Seq libraries was performed on a HiSeq 2500 instrument (Illumina) with 100 bp (x2) reads according to manufacturer's instructions. 36 libraries with distinct barcodes were sequenced in 6 lanes (3 flow cells). The depth of RNA-seq was 15 million reads per sample.