GSM6751697
Chromatin RNA extraction was carried out as previously described (Chen et al., 2019) with  some modifications. Briefly, cells were seeded in 15-cm dishes and were treated with JTE-607 when cell confluency reached 75-80%. Adherent cells were wash with 10 ml of ice-cold PBS twice, scraped from the dishes, and collected into a 10-ml tube. Suspended cells were collected into a 10-ml tube, centrifuged at 420x g at 4°C for 5 min, and then washed with 10 ml ice-cold PBS twice. After removal of PBS, cells were re-suspended in 4 ml of ice-cold HLB+N buffer, containing 10 mM Tris- HCl (pH 7.5), 10 mM NaCl, 2.5 mM MgCl2 and 0.5% (v/) NP-40, and were left on ice for 5 min. Cells were then underlaid with 1 ml of ice-cold HLB+NS buffer (10 mM Tris-HCl (pH 7.5), 10 mM NaCl, 2.5 mM MgCl2, 0.5% (v/v) NP-40 and 10% (wt/vol) sucrose), and were centrifuged at 420x g at 4°C for 5 min. After careful removal of the supernatant, nuclear pellets were re-suspended in 125 ml of ice-cold NUN1 buffer, containing 20 mM Tris-HCl (pH 7.9), 75 mM NaCl, 0.5 mM EDTA, and 50% (v/v) glycerol, followed by addition of 1.2 ml of ice-cold NUN2 buffer, containing 20 mM HEPES-KOH (pH 7.6), 300 mM NaCl, 0.2 mM EDTA, 7.5 mM MgCl2, 1% (v/v) NP-40, and 1 M urea. Samples were vortexed at the maximum speed, followed by incubation on ice for 5 min. Chromatin pellets were collected after centrifugation at 16,000x g at 4°C for 2 min and were re-suspended with 50 ul RNase-free water. The chromatin pellet was dispersed by using a 200 µl tip before chromatin RNA was extracted with 1 ml TRIzol. Chromatin RNA was subject to ribosomal removal  before cDNA library construction by using the NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina Sequencing were carried out on an Illumina HiSeq machine at Ademera Health (South Plainfield, NJ, USA).