GSM6732810: K562 WT_rep2; Homo sapiens; RNA-Seq
Four days after virus infection and puromycin selection, cells were collected for Ago2 IP using Ago2 antibody (Abnova) and the RNA products were purified using Trizol and PAGE-size selected for the intermediate fragments between 40 nt and 80 nt. Isolated RNAs are ligated to 3' adapter using T4 RNA Ligase 2 (truncated KQ) and then purified for reverse transcription by barcoded primer containing complementary sequences to the 3' adaptor on the RNA. Following, the cDNA products are purified on PAGE and circularized using CircLigase II, annealed to the Cut Oligo and digested using BamHI. Finally, the library is amplified from the linear cDNAs using Phusion High-Fidelity PCR master mix with P5/P3 Solexa primers for 20 cycles. The PCR products are purified on Novex 8% TBE gel and send for sequencing with HiSeq 4000 in PE100 mode.