GSM5533428: Control_Flag_1; Homo sapiens; RIP-Seq
After cross-link with 0.75 % formaldehyde, the cells were collected by trypsinization, lysed in 1 ml M-PER buffer (78501, Thermo Fisher Scientific) with 100 U/ml RNase inhibitor and 1 × protease inhibitor on ice for 20 min, and sonicated in a Bioruptor Pico device (Diagenode) with 30s on/30s off for 10 cycles. Then the lysates were collected and subject to RIP with Flag antibody and Protein A/G Magnetic Beads. The RIP RNAs were recovered from the beads with RNeasy mini kit (74104, Qiagen). Both Input and RIP RNAs were used for sequencing. KAPA Stranded mRNA-Seq Kit (Illumina Platforms) was utilized to construct library and sequencing was performed on an Illumina Hiseq 2500.