GSM7842890: UT_R1; Mus musculus; RNA-Seq
Chromatin-associated RNA was extracted as described (Comoglio et al., 2019). Briefly, 5-15x106 BMDM cells were lysed with ice-cold 10 mM Tris-HCl (pH 7.5), 0.05% NP-40, and 150 mM NaCl buffer for 5 min. The lysate was then layered on 2.5 vol of a chilled sucrose cushion and centrifuged at 14,000 rpm for 10 min at 4°C. The nucleus pellet was gently washed with ice-cold PBS/1 mM EDTA and then resuspended in a 20 mM Tris-HCl (pH 7.9), 75 mM NaCl, 0.5 mM EDTA, 0.85 mM DTT, 0.125 mM PMSF, and 50% glycerol buffer by gentle flicking of the tube. An equal volume of a cold 10 mM HEPES (pH 7.6), 1 mM DTT, 7.5 mM MgCl2, 0.2 mM EDTA, 0.3 M NaCl, 1 M urea, and 1% NP-40 buffer was added. The tube was gently vortexed and then centrifuged at 14,000 rpm for 2 min at 4°C. The chromatin pellet was then dissolved in TRIzol (Invitrogen). Chromatin-associated RNA was purified with TRIzol with an additional phenol/ chloroform extraction step prior to precipitation. We used 60-100 ng of the nascent RNA for cDNA-library synthesis using the TruSeq Stranded Total RNA Sample Preparation kit (Illumina RS-122-9007) with the ribosomal depletion step. Libraries were quantified with the Quantifluor reagent (Promega cat. E2670) and the quality of the size was controlled with the Tapestation instrument (Agilent) using the high-sensitivity assay HD5000 (Agilent cat. 5067-5592). cDNA libraries were sequenced on an Illumina NovaSeq platform with 51-bp paired-end reads.