GSM9599301: IgG_biol rep 2; Homo sapiens; RIP-Seq
IP lysis buffer (25 mM Tris-HCl pH7.5, 150 mM NaCl, 1 mM EDTA, 1% NP-40, and 5% glycerol with phosphatase inhibitors (2mM of sodium fluoride, sodium orthovanadate, b-glycerophosphate, 1mM of sodium pyrophosphate), 1mM DTT, 1mM PMSF , 1X protease inhibitor cocktail (Sigma), and RNase-OUT (Invitrogen)). Cells were lysed and 10% of the lysate for each sample was taken as input. The remaining lysates were used for IP. The cleared lysate was immunoprecipitated using protein-A magnetic beads (NEB) complexed with SECISBP2 antibody (12798-1-AP, Proteintech) or control IgG for 2 hours at 4 degrees celcius. RNA for each sample was extracted using TRIzol/Chloroform. Extracted RNA was treated with DNase (Invitrogen) and purified using Qiagen RNeasy Kits. Extracted RNA samples were reverse transcribed into complementary DNA (cDNA) and amplified according to the SMART-Seq2 protocol (PMID: 24385147). The cDNA libraries were fragmented and barcoded using Nextera XT DNA Library Preparation Kit (Illumina). The libraries were sequenced on the NovaSeq X Plus platform (Illumina).