GSM6065758: HEK-293T cells, HA-Dicer/pFlag 2; Homo sapiens; RIP-Seq
HEK 293T cells were transfected with HA-Dicer/pFlag, HA-Dicer/Flag-Gag, HA/Flag-Gag (negative control). After 48 h, cells were washed once with PBS and lysed with RNA extraction buffer (150 mM NaCl, 50 mM Tris, pH 7.5, 5 mM EDTA, 0.5% Nonidet P-40, 0.5% of RNaseOUT (Invitrogen), 0.2% of Vanadyl (NEB), 100mM of DTT (Invitrogen) protease and phosphatase inhibitor cocktail (Roche)). 30 µl of agarose protein A beads (Millipore) washed with RNA extraction buffer were added to 1 mg of protein lysate for a 1.5 h incubation at 4 ℃ on a rocking platform. After centrifugation, the supernatant was incubated with 50 µl of 1% BSA blocked Pierce anti-HA magnetic beads (Thermo Fisher) or 1% BSA blocked Pierce NHS-activated magnetic beads (Thermo Fisher) conjugated with a mouse monoclonal IgG1 isotype antibody (Thermo fisher)   overnight at 4 ℃  in constant rotation. Next day, samples were magnetically separated and the flow through was discarded. The magnetic beads were washed 3 times with RNA extraction buffer and separated in two samples for protein and RNA analysis. For protein analysis, 30 µl of Laemmli sample buffer was added to the beads and incubated 5 min at 95 °C. RNA extraction was performed on the second sample of the beads after adding Trizol reagent and purified with miRNeasy mini kit (QIAGEN). An RNase-free DNase (QIAGEN) was used according to kit protocols in the miRNeasy columns to eliminate possible DNA contamination. Libraries were synthesized from 300 ng of total RNA with the New England BioLabs NEBNext® Small RNA Library Prep Kit for Illumina®. Libraries were run in an Illumina HiSeq2500 SR50 sequencing lane at Genome Quebec, Canada. RIP-seq-small RNAs