GSM8565508: ZFC3H1_FKBP12^F36V, 11A clone, dTAG 26h; Mus musculus; OTHER
Chromatin RNA was extracted from one confluent T25 plates of mESCs. Briefly, cells were trypsinised and washed in PBS. Cells were lysed on ice in RLB (10 mM Tris pH 7.5, 10 mM KCl, 1.5 mM MgCl2, and 0.1% NP40), and nuclei were purified by centrifugation through a sucrose cushion (24% sucrose in RLB). The nuclei pellet was resuspended in NUN1 (20 mM Tris pH 7.5, 75 mM NaCl, 0.5 mM EDTA, 50% glycerol), then lysed with NUN2 (20 mM Hepes pH 7.9, 300 mM, 7.5 mM MgCl2, 0.2 mM EDTA, 1 M Urea). Samples were incubated for 15 minutes on ice then centrifuged at 2800 g to isolate the insoluble chromatin fraction. The chromatin pellet was resuspended in TRIzol by passing multiple times through a 23 gauge needle. Finally chromatin-associated RNA was purified through standard TRIzol/chloroform extraction followed by isopropanol precipitation. Samples were then treated with Turbo DNAse. Approximal 200 - 500 ng of RNA was used for library preparation using the Illumina TruSeq stranded total RNA kit. Libraries were quantified by qPCR with KAPA Library Quantification DNA standards (KAPA Biosystems). The libraries were pooled and 2X 81 paired end sequencing was performed using Illumina NextSeq 500/550.