GSM6421800: ASC48_LACE_seq_bio1_tech2; Mus musculus; RIP-Seq
YTHDC1 antibody (Abcam, ab122340) was used to pull down specific protein-RNA complex from lysate.MNase was used to cut RNAs into YTHDC1-associated short fragments on beads. The 3' ends of fragmented RNAs were then dephosphorylated and ligated with a 5' pre-adenylated linker containing four randomized nucleotides. Biotinylated primer containing the T7 promoter was used for reverse transcription which stopped at crosslinking site (YTHDC1 binding site). cDNAs were poly(A) tailed and purified with streptavidin beads. Second-strand cDNAs were synthesized on beads with an adaptor containing oligo-(dT). After a pre-amplifying step by PCR, T7 RNA polymerase was used to amplify trace amounts of truncated cDNAs linearly. Then the products were PCR converted into libraries for single end sequencing on Illumina platform (Nextseq 550)