GSM6923099: TOTAL, rep2; Mus musculus; RNA-Seq
Freshly dissected testes were collected in ice-cold PBS and homogenized in Lysis buffer containing 100 mM KCl, 20 mM Tris-HCl pH 7.5, 2 mM MgCl2, 0.3% NP-40, Halt protease and phosphatase inhibitors (Thermo Fisher), RNasin Ribonuclease Inhibitor (Promega) in nuclease-free water. Lysates were clarified by centrifugation, supernatants were recovered and EDTA was added at a final concentration of 10 mM. Extracts (TOTAL) were further fractionated by 10-50% sucrose gradient ultracentrifugation; fractions 5, 6, 7 and 8 were pooled and used as INPUT for either anti-L1 ORF1p co-immunoprecipitations (IP) or incubations with Beads Only (BO). RNA from TOTAL, INPUT, IP and BO samples was purified using TRIzol reagent and treated with TURBO DNase (Invitrogen). Libraries were generated using the TruSeq Stranded Total RNA kit (Illumina), with Ribo-Zero rRNA depletion.