GSM5696890: rna_nt_rep2; Mus musculus; RNA-Seq
RNA: Total RNA was isolated from primary mouse hepatocytes using NucleoSpin kit (Macherey-Nagel cat# 740955.25) according to the manufacturer's protocol. ChIP: cells were cross-linked with 1% formaldehyde for 10 min at room temperature and quenched with 0.125M glycine. Crosslinked samples were washed in PBS, resuspended in ChIP lysis buffer (0.5% SDS, 10mM EDTA, 50mM Tris-HCl pH8) and sonicated (Bioruptor, Diagenode) to release 100-1000 bp fragments. Antibodies (4 μg per 300 μg chromatin) against H3K27ac (Active motif #39133) or GR (CST #3660) were conjugated to magnetic beads (Sera-Mag, Merck #GE17152104010150) for 2 h at 4°C. Chromatin was immunoprecipitated with antibody-bead conjugates over night at 4°C. Immunocomplexes were washed sequentially with the following buffers: low-salt buffer (0.01% SDS, 1% Triton x-100, 2 mM EDTA, 20mM Tris-HCl pH8, 150mM NaCl), high salt buffer (0.01% SDS, 1% Triton x-100, 2mM EDTA, 20mM Tris-HCl pH8, 500mM NaCl), low salt buffer and TE buffer (10mMTris-HCl, 1mM EDTA pH8). Chromatin was de-proteinized with proteinase K (Hy Labs EPR9016) for 2 h at 55°C and de-crosslinked over night at 65°C. DNA was subsequently phenol-chloroform purified and ethanol precipitated For quality control of RNA yield and library synthesis products, the RNA ScreenTape and D1000 ScreenTape kits (both from Agilent Technologies), Qubit® RNA HS Assay kit, and Qubit® DNA HS Assay kit (both from Invitrogen) were used for each specific step. mRNA libraries were prepared from 1 µg RNA using the KAPA Stranded mRNA-Seq Kit, with mRNA Capture Beads (KAPA biosystems, cat# KK8421). ChIP DNA libraries were prepared using the KAPA HyperPrep Kit (KAPA biosystems, cat# KR0961).