GSM4869207: Cel pull-down: IGROV1-R10 RNA purified together with control miRNA; Homo sapiens; RNA-Seq
Cells were pelleted and washed once in cold PBS. Cells were then resuspended in 700 µl lysis buffer (20 mM Tris-HCl pH 7.5, 5 mM MgCl2, 100 mM KCl, 0.3% NP-40, protease inhibitor, RNase inhibitor 60 U/ml (RNase OUT, ThermoFisher Scientific)) and incubated on ice for 5 min. Samples were then centrifuged (4°C, 13,000 g) to remove cell debris and nucleus. 10 % of the lysate was sampled and submitted to RNA extraction with Trizol reagent, to be later used as “input”, while the rest was incubated on 50 µl magnetic streptavidin beads (Dynabeads MyOne Streptavidin T1, ThermoFisher Scientific) at 4°C during 4 h under agitation. Before this incubation step, beads were washed twice and blocked by incubation in lysis buffer supplemented with BSA (1 mg/ml) (Promega) and yeast tRNA (1 mg/ml) (Sigma-Aldrich) at 4°C during 2 h under agitation. At the end of the 4 h incubation period with lysate, beads are washed 5 times in 500 µl lysis buffer. After final wash, they are resuspended in Trizol reagent to proceed with RNA purification. TruSeq Stranded Total RNA Sample Prep' Illumina