GSM9440840: mRNA_RIPseq_N2_antiGFP_rep3; Caenorhabditis elegans; RIP-Seq
At the time of sample collection, animals were harvested, washed three times with M9 buffer, once with 0.1 M KCl and dry pellets were snap frozen in liquid nitrogen. Upon thawing, the pellets were lysed in extraction buffer (50 mM HEPES/KOH (pH 7.4 at 4°C), 150 mM KCl, 5 mM MgCl2, 0.1% (v/v) Triton X-100, 10% (w/v) glycerol, 1 mM PMSF, 1 tablet/10 ml cOmplete Mini Protease Inhibitor Tablets (EDTA-free; Roche, 11836170001), 200 U/ml RNaseOUT Recombinant Ribonuclease Inhibitor (Thermo Fisher Scientific, 10777019)), with mortar and pestle in the presence of liquid nitrogen. Anti-GFP IPs were performed by incubating 3 mg total protein with 50 µl of Dynabeads Protein G (Thermo Fisher Scientific, 10003D) bound to anti-GFP antibody (5 µg/IP; Abcam, ab290). The immunoprecipitated RNA was extracted by directly adding TRI Reagent (MRC, TR-118) to the beads and snap-freezing in liquid nitrogen. For each condition (N2 and HW2805), three IPs were performed in parallel as technical replicates. Three aliquots of input lysate each corresponding to 100 µg total protein were snap-frozen in liquid nitrogen upon addition of TRI Reagent for extraction of the input RNA. RNA was extracted using Direct-zol RNA Microprep kit (Zymo Research; R2062) according to the manufacturer's protocol including the on column DNase digestion. RNA was eluted in nuclease-free water (Thermo Fisher Scientific, Ambion, AM9937). Illumina Stranded mRNA-seq
GEO
GSM9440840_r1