GSM4970392: meCLIP_MCF7_rep2_INPUT; Homo sapiens; RIP-Seq
Cells were cultured in appropriate media and total RNA was isolated using the Trizol (15596018, Invitrogen). Poly(A) selection was performed using the Magnosphere® Ultrapure mRNA Purification Kit (9186, Takara). RNA was fragemented at 70C for ~3min using RNA Fragmentation Reagents (AM8740, Ambion). A small amount (~500ng) of fragmented RNA was saved for use as the input sample. Library was prepared using eCLIP-seq protocol with minor modifications. Briefly, the fragmented poly(A) RNA was incubated with anti-m6A antibody (Abcam Ab151230) and then crosslinked twice at 150 mJ/cm2 (254nm wavelength) using a Stratalinker UV Crosslinker. The RNA:antibody sample was incubated with Protein A/G Magnetic Beads (88803, Pierce) overnight at 4C and then a RNA adapter is ligated on the 3' end of the sample. The RNA:antibody complex is then ran on an SDS-PAGE gel and transferred to a nitrocellulose membrane to remove any non-crosslinked RNA. Following treatment with Proteinase K to remove nearly all of the antibody except the crosslinked amino acid, the RNA is isolated, one adapter is ligated, and the RNA is converted into cDNA for full library amplification. Sequencing of the cDNA libraries was primarily performed using an Illumina NovaSEQ 6000 to generate 2x150bp paired-end runs consisting of 40 million raw reads per sample.