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| Status |
Public on Apr 08, 2021 |
| Title |
Corticosterone RNA Pol2 (pSer2) ChIP - GH2012_constant_180_rep2 |
| Sample type |
SRA |
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| Source name |
Liver
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| Organism |
Rattus norvegicus |
| Characteristics |
strain: Sprague Dawley
Sex: Male
tissue: Liver
treatment: Corticosterone infusion - constant - 180min
chip antibody: 2 ug ab5095 (Abcam, UK)
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
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)
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
ChIP-Seq
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| Data processing |
Basecalling RTA 1.18.61 Illumina instrument run time analysis software
Adapters removed by Trimmomatic-0.36, -phredd33 SE
Aligned using bowite2 (default parameters)
Duplicate tags removed by samtools (default parameters) and normalised to a total of 10 million by makeTagDirectory -single -genome rn6 -checkGC (HOMER v4.9.1)
Approximate ChIP-fragment densities compiled for visualisation purposes (i.e. UCSC genome browser) for each replicate by makeUCSCfile (HOMER v4.9.1)
Enrichment of GR tags to 1 % input control, were identified by findPeaks (HOMER v4.9.1) using relaxed settings -F1 -L1 -P.1 -LP.01 -poisson .1 -style factor. Replicate concordance was assessed using an irreproducible discovery rate (IDR v2.0.3) set to 0.01. Overlapping enrichments between replicates were merged using mergePeaks (HOMER v4.9.1) and filtered according to the IDR estimated confidence threshold. Overlapping confident enrichment regions were merged again (mergePeaks) to create a single list of enriched GR locations across conditions.
pSer2 Pol2 enrichment regions were manually restricted to transcript coding regions larger than twice the fragment length (> 320b) and limited to 10 Kb from the transcriptional start site (TSS) based upon Ensemble release Rnor_6.0.92 co-ordinates.
Raw tags counted within GR and pSer2 Pol2 regions by annotatePeaks.pl -raw (HOMER v4.9.1)
Input tags subtracted and differential enrichment identified by DESeq2 analysis by getDifferentialExpression.pl -AvsA -log2fold 0 (HOMER v4.9.1) (n=2)
Input tags subtracted and differential enrichment identified by DESeq2 analysis by getDifferentialExpression.pl -AvsA -log2fold 0 -norm2total (HOMER v4.9.1) (n=2)
List of IDR filtered enriched regions after 140 min (PLS_GR_140_peaks.txt) and 180 min (PLS_GR_180_peaks.txt) pulsatile CORT infusion, 140 min (CNS_GR_140_peaks.txt) and 180 min (CNS_GR_180_peaks.txt) constant CORT infusion, 140 min and 180 min VEH infusion (VEH_GR_peaks.txt) and resultant merged regions from all treatments (GR_peaks.txt). Approximate ChIP-fragment densities are compiled for visualisation purposes (i.e. UCSC genome browser) for each replicate (all bedGraph.gz files). Final matrix includes peak ID (column 1), GR enrichment locations (columns 2-5), whether a region was enriched to input control by each infusion condition (column 6-10),normalised tag counts (columns 27-36) and DESeq2 results, formatted by log2 fold change, Wald test p-value, Benjamin-Hochberg adjusted p-value and wether an enriched peak was detected for either compared treatment (columns 37-75) (GRChIP_allresults.txt).
Enrichment regions were manually restricted to transcript coding regions larger than twice the fragment length (> 320b) and limited to 10 Kb from the transcriptional start site (TSS) based upon Ensemble release Rnor_6.0.92 co-ordinates (pSer2Pol2_intragenic_regions.txt). Approximate ChIP-fragment densities are compiled for visualisation purposes (i.e. UCSC genome browser) for each replicate (all bedGraph.gz files). Final matrix includes gene ID (column 1), chr pSer2 Pol2 enrichment locations and strand (columns 3-6), normalised tag counts (columns 22-31) and DESeq2 results, formatted by log2 fold change, Wald test p-value and Benjamin-Hochberg adjusted p-value (columns 32-61) (pSer2Pol2ChIP_allresults.txt).
Genome_build: rn6
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| Submission date |
Apr 07, 2021 |
| Last update date |
Apr 08, 2021 |
| Contact name |
Benjamin P Flynn |
| E-mail(s) |
ben.flynn@bristol.ac.uk
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| Organization name |
University of Bristol
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| Street address |
Dorothy Hodgkin Building, Whitson street
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| City |
Bristol |
| State/province |
County (optional) |
| ZIP/Postal code |
BS13NY |
| Country |
United Kingdom |
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| Platform ID |
GPL14844 |
| Series (1) |
| GSE171647 |
Corticosterone pattern-dependent glucocorticoid receptor binding and transcriptional regualtion |
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| Relations |
| BioSample |
SAMN18649202 |
| SRA |
SRX10533683 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5229685_GH2012_CNS_POL_180.ucsc.bedGraph.gz |
48.8 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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