GSM2497656: rep2_AI_EZH2; Homo sapiens; RIP-Seq
Cells were irradiated once for 400 mJ/cm2. Take up crosslinked cells in ice-cold lysis buffer, then add RQ I DNaseat incubated at 37 °C for 3 min at 1,000g. The mixture was then centrifuged at 10,000 x g at 4 °C for 10 min to remove cell debris. The supernatant was incubated with DynaBeads protein G conjugated with anti-EZH2 antibody (source: Millipore, cat# 17-662, lot# 2366583) or IgG (source: Millipore, cat# 17-662, lot# 2366583) at 4 °C for 2 hours. The beads were washed with Wash buffer, High-salt Wash buffer and 1X PNK Buffer, respectively. Resuspend the beads in dephosphorylation mix and wash the beads 3 times with 1X PNK Buffer. Then resuspend the beads in phosphorylation mix and wash the beads 2 times with 1X PNK Buffer. Resuspend the beads in Elution Buffer. The supernatant was used to run gels. A region 30 kDa above the protein size is excised. Add PK buffer (900 µl) and 10mg/ML proteinase K (100 µl) to the membrane pieces, the isolated RNA was used for prepare sequencing library. For high-throughput sequencing, the libraries were prepared following the manufacturer's instructions and applied to illumina NextSeq 500 system for 151 nt pair-end sequencing and Illumina Genome Analyzer Iix for single-end sequencing by ABlife. Inc (Wuhan, China).
GEO Accession
GSM2497656