GSM6531648: batch1_rip_seq_Setd2_KO2_IP_rep1; Mus musculus; RIP-Seq
Cells were collected and washed with cold PBS once. Hela cells were spiked in as the internal control for normalization. 1 ml cold polysome lysis buffer (50 mM KCl, 25 mM Tris pH 7.4, 5 mM EDTA, 0.5% NP-40, 0.5 mM DTT, 100 U/ml RNAase inhibitor, and 1× protease inhibitor cocktail) was added and incubated on ice for 10 min. Samples were cleaned by centrifugation at 14,000 rpm 4 °C for 10 min. 10% of the lysate was saved as the input sample. Others were incubated with Anti-DYKDDDDK G1 Affinity Resin beads (GenScript, Cat. #L00432) which were washed with NT2 buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1 mM MgCl2, and 0.05% NP-40) at 4 °C for 4h. Beads were washed with NT2 buffer 5 times to remove unbound material. RNA was purified by Trizol reagent (Sangon Biotech, Cat. #B610409) library preparation was done by RNA-seq library prep kit (VAHTS, Cat. #NR604)