GSM5229676: Vehicle GR ChIP - GH1992_pulsatile_140; Rattus norvegicus; ChIP-Seq
ChIP-Seq  Dissection and fixing  Liver was dissected and 0.4 g fixed in 1 % (v/v) formaldehyde (Sigma, UK), phosphate buffered saline (1.37 M NaCl, 2.68 mM KCl, 10.14 mM Na2HPO4, pH 7.4) solution for 10 min at room temperature. Formaldehyde cross-linking was quenched with addition of glycine (final conc. 125 mM) for 5 min and washed three times in ice cold phosphate buffered saline supplemented with 2 mM NaF, 0.2 mM sodium orthovanadate and 1X cOmplete protease inhibitor (Roche Diagnostics). Fixed liver was stored at -80 °C in 0.2 g samples in 500 µl of S1 Buffer (10 mM HEPES, pH 7.9, 10 mM KCl, 15 mM MgCl2, 0.1 mM (EDTA), pH 8) supplemented with 0.5 mM Dithiothreitol and 2 mM NaF, 0.2 mM sodium orthovanadate and 1X cOmplete protease inhibitor.   ChIP assay  Samples were thawed slowly on ice and adjusted to a final volume of 1 ml/ sample with supplemented S1 buffer and Dounce homogenised. Lysate was centrifuged at 4000 rpm (4 °C) and lysed in supplemented (2mM NaF and 0.2 mM sodium orthovanadate and 1X cOmplete protease inhibitor) sodium dodecyl sulphate (SDS) lysis buffer (2 % SDS, 10 mM EDTA, 50 mM Tris-HCl (pH 8.1)). Chromatin was sonicated using a Branson Sonifier 450 (Branson Ultrasonics, Danbury, CT, USA) to 300-500 bp fragments with multiple 10-sec bursts at 10 % output and centrifuged at 13,000 rpm at 4 °C to remove cellular debris from the chromatin suspension. Sheared chromatin was stored at -80 °C.  ChIP buffers were prepared in house, as described in the EZ ChIP kit protocol (Upstate Biotechnology, Lake Placid, NY, USA) with some modifications for use with animal tissue (as described where relevant). Sheared chromatin was removed from -80 °C storage and thawed slowly on ice, diluted to 50 µg in 100 µl of SDS lysis buffer and 0.9 ml with supplemented (2mM NaF, 0.2 mM NaVan and 1X cOmplete protease inhibitor) ChIP dilution buffer (0.01 % SDS, 1.1 % Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl pH 8.1, 167 mM NaCl). Inputs were immunoprecipitated against either a GR antibody cocktail (2 µg of PA1-510A, 4 µl of PA1511A (Thermo Fisher, USA) and 4 µg of M-20X sc-1004X (Santa Cruz, USA)) or the serine 2 phosphorylated RNA polymerase II complex (2 µl ab5095 (Abcam, UK)). Rabbit IgG antibodies were used as negative control (2 µg of sc-2027 (Santa Cruz, USA)). Inputs were incubated overnight at 4 °C and incubated with protein A Dynabeads for 4 hrs (Sigma-Aldrich, UK). To reduce non-specific binding, the DNA-Antibody-Dynabead slurries were sequentially washed with 150 mM salt buffer (0.1 % SDS, 1 % Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.1), 500 mM salt buffer (0.1 % SDS, 1 % Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.1), LiCl buffer (0.25 M LiCl, 1 % IGEPAL-CA630, 1 % deoxycholic acid sodium salt, 1 mM EDTA, 10 mM Tris-HCl pH 8.1) and twice in TE buffer (10 mM Tris-HCl, 1 mM EDTA) (pH 8.0). Complexes were eluted from the Dynabeads in 1 % SDS 100 mM and 0.1M NaHCO3. NaCl was added (300 mM final concentration) and crosslinks reversed overnight at 65 °C. RNA was removed using RNase treatment (Roche Diagnostics) at 65 °C and protein was digested using proteinase K (Ambion, Huntington, UK) after adjusting each solution with EDTA (1 mM final) and Tris-HCl (4 mM final) at 45 °C. DNA was extracted using 25:24:1 phenol-chloroform-isoamyl alcohol (Sigma, UK) followed by 24:1 chloroform-isoamyl alcohol (Sigma, UK). DNA in the aqueous phase was precipitated overnight at -20 °C in 2.5 VOL 100 % ethanol and 20 µg glycogen (Sigma-Aldrich, UK). DNA was pelleted by centrifugation at 13,000 rpm, 4 °C and washed in 70 % Ethanol (13,000 rpm, 4 °C), air dried and suspended in 40 µl nuclease free water (Ambion, Huntington, UK).  For each ChIP-seq replicate/condition/timepoint, liver samples from 6 rats were collected (72 rats in total). From each replicate/condition/timepoint, 10 x 100ug chromatin aliquots were prepared and immunoprecipitated with antibodies to either GR or pSer2 Pol2 before pooled, concentrated using a UniVapo Vacuum concentrator (Transcriptomics facility, UOB) to approx. 35 µl and sequenced. Note: Two separate ChIP-seq experiments were performed independently to allow for concordant peak calling analysis as previously described ChIP-Seq  Library prepared according to TrueSeq SBS v3-HS kit protocol (Illumina)