title: GSM2850142: RIP_lysate_IP2; Mus musculus; RIP-Seq
library_construction_protocol: For RIP-Seq：Cells were washed twice with cold PBS and the cell pellet was re-suspended with 2 volumes of lysis buffer (150 mM KCl, 10 mM HEPES pH 7.6, 2 mM EDTA, 0.5% NP-40, 0.5 mM DTT, 1:100 protease inhibitor cocktail, 400 U/mL RNase inhibitor).For RNA-Seq: Total RNA was extracted by Trizol. For RIP-Seq：Lysate was incubated on ice for 5 min centrifuged for 15 min to clear the lysate. 1/10 volume of cell lysate was saved as input and total RNA was extracted by trizol. The rest of cell lysate was incubated with 5ug anti-YTHDF1(Proteintech) at  4°C overnight with gentle rotation followed by incubation with 40ul protein G beads for 1 hour at 4°C. The beads were then washed five times with 1 mL ice-cold washing buffer (200 mM NaCl, 50 mM HEPES pH 7.6, 2 mM EDTA, 0.05% NP-40, 0.5 mM DTT, 200 U/mL RNase inhibitor). The IP complex was re-suspended in 400μl 1xProteinase K and digested with 2 mg Proteinase K at 55°C for 1 hour. RNA was then extracted by RNA isolation kit (Zymo). Input and IP RNA of each sample were used to generate the library using TruSeq stranded mRNA sample preparation kit (Illumina). For RNA-Seq: Extracted RNA of each sample were used to generate the library using TruSeq stranded mRNA sample preparation kit (Illumina).