title: GSM3400098: SART-3_input_1; Caenorhabditis elegans; RIP-Seq
library_construction_protocol: Lysis of pellets of about 1 ml with L3/L4 stage worms was performed 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, 7 mg/ml cOmplete Protease Inhibitor Tablets (EDTA-free), 200 U/ml SUPERase In RNase Inhibitor), by adding liquid nitrogen and grinding with mortar and pestle. All subsequent steps were performed at 4 °C. Lysates were centrifuged at 12,000g for 10 min and the supernatant used for Anti-FLAG IPs. 3 mg total protein were incubated with 30 µl of anti-FLAG M2 magnetic beads (Sigma–Aldrich; M8823) for 3 h on a rotating wheel. With extraction buffer without protease and RNase inhibitors, the beads were washed 5 times for 5 min. Finally, the bound RNA was extracted by directly adding Tri Reagent to the beads. 5 IPs were performed in parallel for each condition to recover a sufficient amount of RNA. Using an aliquot of remaining input lysate, input RNA was extracted with Tri Reagent. TruSeq Stranded mRNA HT Sample Prep Kit (Illumina; RS 122 2103)
attributes.tag: GEO Accession
attributes.value: GSM3400098