GSM3624484: SETD5+/-_T10_rep1; Mus musculus; RNA-Seq
Cells were lysed in HLB/NP40 buffer (10 mM Tris-Hcl, pH 7.5, 10 mM NaCl, 0.5% NP40, and 2.5 mM MgCl2) 5 minutes in ice. Nuclei were isolated by under-laying HLB/NP40/Sucrose buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 0.5% NP40, 2.5 mM MgCl2, and 10% Sucrose) and centrifuging at 1400 g for 5 minutes at 4°C. The chromatin fraction was isolated by resuspending the nuclei in 125 μl of NUN1 solution (20 mM Tri-HCl, pH 8.0, 75 mM NaCl, 0.5 mM EDTA, 50% Glycerol and proteinase inhibitor) followed by 1.2 ml NUN2 buffer (20 mM HEPES-KOH pH 7.6, 7.5 mM MgCl2, 0.2 mM EDTA, 300 mM NaCl, 1 M Urea, 1% NP40, proteinase and phosphatase inhibitor cocktail), incubating on ice for 15 minutes and centrifuging at 15000 g for 10 minutes at 4°C. Chromatin-bound RNA was extracted with Trizol reagent (Thermo Fisher cat. #15596026). DNA and ribosomal RNA contaminants were removed by DNase (Qiagen cat. #79254) and RiboZero (Illumina cat. # MRZG12324) treatments, respectively. RNA quality and concentration were assed at the 2100 Bioanalyzer (Agilent cat. #G2939BA) and the Qubit fluorometer (Thermo Fisher cat. # Q33226), respectively. Chrom-RNA-seq libraries were prepared by using the stranded Small RNA Library Prep Kit (Illumina cat. # RS-200-0012), with minor modifications and sequenced as single reads of 50 bp with the Illumina HiSeq2500. Three independent biological replicates were performed for each cell line and time point and sequenced as independent libraries.
GEO Accession
GSM3624484