GSM7061505: K562 cells, SF3B1 K700E, 4 days, replicate 2 [nascentSRSseq_K562_SF3B1K700E_Replicate2]; 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 The isolated polyA, rRNA depleted RNA was then fragmented with a Bioruptor UCD-200, and the fragmented RNA was used to produce barcoded cDNA sequencing libraries using the SMARTer® Universal Low Input RNA Kit