GSM7743924: AGO IP, Dicer N1, SINV-GFP MOI 0.02 24 hpi, rep2; Homo sapiens; Sindbis virus; miRNA-Seq
Cells were collected in 1.3 mL of ice-cold lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM EDTA, 1 % NP-40, 0.5 mM DTT, 0.1 U/mL of RNase inhibitor and protease inhibitor cocktail (complete Mini, Merck, Sigma Aldrich)). Cells were lysed by putting the extract on ice for 15 min. First, human AGO proteins were immunoprecipitated from whole extract to enrich for loaded small-RNAs. AGO-IP was done following the protocol described in (Hauptmann et al., 2015). Briefly, 50 µL of magnetic Dynabeads coupled to G protein (Invitrogen, Fisher Scientific) were coupled to anti-Flag M2 antibody (F1804, Merck, Sigma Aldrich) overnight at 4°C under rotation. The day after, Flag-TNRC6B WT or mutant (with Tryptophans mutated to Alanines; called TNRC6B Ala) peptides were incubated with the coupled beads for 3 h at 4°C under rotation. Meanwhile, lysates were cleared with a centrifugation step at 10 000 g for 10 min at 4°C and 100 µL were kept as INPUT (20 µL for proteins and 80 µL for RNAs). Then, lysates were divided in two and put on the beads coupled to the peptides and incubated at 4°C for 3 h under rotation. After 4 washing steps with 300 µL of ice-cold washing buffer, beads were split: 280 µL were eluted with 1 mL of TRI Reagent solution (Invitrogen, Fisher Scientific) and RNAs were isolated according to the manufacturer's instructions Illumina TruSeq small RNA library preparation kit was used according to the manufacturer's protocol (RS-200-0012, Illumina).