GSM7060925: C4-2 Cell line EZH2_input_rep2; Homo sapiens; RIP-Seq
Total RNA (n=4 for siCTRL, n=4 for siEZH2, n=6 for siADAR1 and n=5 for siADAR2) was isolated from C4-2 cells using RNeasy Plus Mini Kit (Qiagen) and subjected to BGI for library preparation and sequencing using the DNBseq platform. Meanwhile, the genomic DNA was extracted from C4-2 cells using Quick-DNA Kit (Zymo Research) and subjected to BGI for human true PCR-free 30x coverage WGS. The eCLIP-seq experiment was performed in duplicates following the method described previously[63]. For each sample, a total of 50 million C4-2 cells were crosslinked, lysed and partially digested by RNase I (Thermo Fisher). The resulting lysates were incubated with the antibody of interested RBP and Protein A/G magnetic beads (Pierce) at 4 ºC overnight, followed by washing, end repair and 3′ adaptor ligation. The protein-RNA complexes were then eluted from the beads, resolved by denaturing gel electrophoresis and transferred to a nitrocellulose membrane. Subsequently, the region of interest was cut from membrane and subjected to RNA extraction, cDNA synthesis and library preparation. The cDNA library was further amplified using Q5 PCR Master Mix (NEB) and sent to BGI for sequencing using the DNBseq platform. For high-throughput sequencing of RNA-seq, DNA-seq, input and CLIP RNA samples were shipped to BGI for library preparation and sequencing using the DNBseq platform.