GSM7676725: HKC_sgVHL_sg2_RNA_Rep3; Homo sapiens; RNA-Seq
Cellular RNA was extracted using the RNeasy mini kit (Qiagen, 74104) according to the manufacturer's instructions. Complementary DNA (cDNA) was synthesized iScript Reverse Transcription Supermix (Bio-Rad, 1708841). RIP was performed using a modified native RNA immunoprecipitation procedure as previously described. In briefly, approximately 3x10^7 HKC cells were lysed with lysis buffer (100mM KCl, 5mM MgCl2, 10mM HEPES pH 7.0, 0.5% NP-40, 1mM DTT, supplemented with RNase inhibitor and EDTA-free Protease inhibitor before using). Incubate the cell lysate with protein-A/G magnetic beads that immobilized with corresponded antibodies for overnight at 4 °C. Wash the beads 6 times with NT-2 buffer (50mM Tirsh-HCl pH 7.4, 250mM NaCl, 1 mM MgCl2, 0.05% NP-40, 20mM EDTA, 1mM DTT, supplemented with RNase inhibitor before using). Input and co-immunoprecipitated RNAs were first digested with Proteinase K buffer (NT-2 buffer supplemented with 1% SDS and 1.2 mg/ml Proteinase K) at 55 °C for 30 min and then recovered by TRIzol according to the supplier's instructions. Immunoprecipitated RNA was extracted with RNeasy mini kit (Qiagen, 74106). cDNA library was prepared with NEBNext® Ultra™ II RNA Library Prep Kit for Illumina (NEB, E7775). Samples were subjected for qPCR analysis or sequenced on the Illumina NextSeq 500 NGS system as 75 bp pair-ended reads.