GSM9512988: SMCHD1_K518E, clone A4, ML day 5 rep1; Mus musculus; RNA-Seq
Chromatin RNA was extracted from one confluent 90mm Petri dish of mESCs. Briefly, cells were trypsinised and washed in PBS. Cells were lysed on ice in HLBN buffer (0.01M NaCl, 2.5mM MgCl2, 10mM Tris pH 7.5, 0.1% NP-40), and nuclei were purified by centrifugation through a sucrose cushion (24% sucrose in HLBN). The nuclei pellet was resuspended in NUN1 (75mM NaCl, 20mM Tris pH 7.9, 0.5mM EDTA, 50% glycerol, 0.1mM DTT) , then lysed with NUN2 (0.3M NaCl, 20mM HEPES pH 7.6, 7.5mM MgCl2, 0.2mM EDTA, 1M urea, 1% NP-40, 0.1mM DTT) . Samples were incubated for 15 minutes on ice and the chromatin fraction was isolated by centrifugation and resuspended in HSB buffer (0.5M NaCl, 10mM Tris pH 7.5, 10mM MgCl2). Samples were treated with DNase and then with 0.2% SDS and 0.4mg/ml proteinase K. Finally chromatin-associated RNA was purified through two rounds of standard TRIzol/chloroform extraction with overnight isopropanol precipitation. Between the two rounds, the samples were treated with DNAse. Approximal 100 - 500 ng of RNA was used for library preparation using the Illumina TruSeq stranded total RNA kit. Libraries were quantified by Qubit. The libraries were pooled and 2X 81 paired end sequencing was performed using Illumina NextSeq 500/550.