GSM7968422: HEK293T shKDM2B, 60min, rep1; Homo sapiens; OTHER
Nuclei was prepared from HEK293T shSCR and shKDM2B cell lines treated by PBS or TNFa. Nuclear run-on assays were performed with Biotin-11-ATP/UTP/CTP/GTP (PerkinElmer). Biotin-labeled RNA was purified by streptavidin beads M-280 (Thermo). The 5′ cap removal and triphosphate repair were performed with RppH (NEB), 5′ hydroxyl repair was then performed with PNK (NEB). 5′ and 3′ adaptor ligation was performed with T4 RNA ligase I (NEB). Reverse transcription was performed with SuperScript III (ThermoFisher). cDNA was amplified using Phusion Hot Start II DNA polymerase (ThermoFisher). DNA Libraries were size selected by PippinHT (Sage) or AMpure XP (Beckman Coulter) and sequenced on Illumina NovaSeq 6000.