GSM7061460: K562 cells, SF3B1 K700E, 4 days, replicate 1 [LRSseq]; Homo sapiens; RNA-Seq
All extraction steps were performed on ice, and all buffers contained 25 uM α-amanitin, 40 U/ml RNase inhibitor, and protease inhibitor. Chromatin fractions were isolated from the cellular pellets using a previously published protocol. Chromatin was immediately dissolved in PBS and TRIzol Reagent. RNA was purified from chromatin using the RNeasy Mini kit according to the manufacturer's protocol, including the on-column DNase I digestion. For genome-wide nascent RNA-seq, samples were depleted three times of polyA(+) RNA using the Magnetic mRNA isolation kit, each time keeping the supernatant, then depleted of ribosomal RNA using oligo probe-based rRNA depletion A DNA adapter was ligated to 3′ ends of nascent RNA using the T4 RNA ligase kit by mixing adapter with nascent RNA. cDNA was generated from the adapter ligated RNA using the SMARTer PCR cDNA Synthesis Kit. cDNA was amplified by 15 cycles of PCR using the Advantage 2 PCR Kit with template switching custom oligo, cleaned up using a AMPure beads