GSM5217223: HEK293T-FH-Ago2 siTRMT6/61A Input TGIRT small RNA rep3; Homo sapiens; ncRNA-Seq
Ago2 RIP was performed in stable FH-Ago2 HEK293T cells. Briefly, 1E7 cells were collected by scraping and washed with DPBS, before lysis in RIP buffer (50 mM Tris-HCl, pH 7.4, 150 mM KCl, 0.5% NP-40 substitute, 0.5 mM DTT, 100X proteinase inhibitor, RNase inhibitor). Cleared lysates were incubated at 4°C for 1 hr with M2 FLAG affinity gel (Sigma #A2220) or anti-MYC mouse antibody 9E10 conjugated on beads as a negative control. After washes, bound RNAs are eluted by Trizol extraction and ethanol precipitation. Small RNA-seq library preparation was performed using NEBNext Small RNA Library Prep Set for Illumina (NEB #7330) with modification to use TGIRT-III RT enzyme (InGex). The resulting amplified libraries were size selected on 8% TBE polyacrylamide gel to enrich for insert of 15-50 nucleotides long RNA.