GSM9207785: Sample_RIP_seq_2_IP; Mus musculus; RIP-Seq
tissues were homogenized with polysome lysis buffer (100 mM KCl, 5 mM MgCl2, 10 mM HEPES (pH 7.2), 0.5% NP40, 1 mM DTT, supplemented with RNase inhibitors and protease inhibitors), and incubated on ice for 5 minutes. After centrifugation at 15,000 g for 15 minutes at 4 ℃, 40μL of supernatant was collected for RNA extraction as input sample and another 40uL was collected for Western blotting analysis. The remaining supernatant was incubated with YTHDF1 antibody-coupled protein A/G beads at 4 °C for 4 h on a rotator. The beads were then washed with ice-cold NT2 buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM MgCl2 and 0.05% NP-40). TRIzol was added to input and IP samples to extract total RNA. RNA quality control was measured on a Qubit. Sample libraries were prepared with SMART-Seq v4 Ultra Low Input RNA Kit (Takara Bio, 63488). Libraries were sequenced on a NoveSeq 6000 (Illumina).