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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Dec 15, 2019 |
| Title |
mESCs, RING1B_DMSO_1 |
| Sample type |
SRA |
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| Source name |
Embryonic Stem Cells
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| Organism |
Mus musculus |
| Characteristics |
cell type: mESC
treatment: 24 h DMSO
cell line: E14TG2a
strain: 129/Ola
chip antibody: anti-RING1B (Cell Signalling D22F2)
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| Treatment protocol |
Mouse ESCs were plated in standard medium at 4 × 104 cells/cm2 and cultured for 24 h. The medium was then replaced with medium supplemented with either EPZ-6438 (BioVision - catalogue no. 2383-5; reconstituted in DMSO) at a final concentration of 2.5µM or DMSO and cultured for a further 24 h prior to harvesting.
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| Growth protocol |
Feeder-free mouse embryonic stem cells (mESCs; E14tg2A - 129/Ola) were cultured on 0.1% gelatin (Sigma - catalogue no. G1890) coated Corning flasks in GMEM BHK-21 (Gibco - catalogue no. 21710-025) supplemented with 10 % foetal calf serum (FCS; Sigma - catalogue no. F-7524), 1,000 units/ml LIF, non-essential amino acids (Gibco - catalogue no. 11140-035), sodium pyruvate (Gibco - catalogue no. 11360-039), 50 μM 2-β-mercaptoethanol (Gibco - catalogue no. 31350-010) and L-glutamine. For passaging, 60–90% confluent ESC culture flasks were washed with PBS, incubated for 2–3 min at RT in trypsin (0.05% v/v; Gibco 25300-054), and tapped to release. Trypsin was inactivated by adding 9 volumes of ESC medium and this mixture was repeatedly pipetted to obtain a single cell suspension. ESCs were centrifuged, resuspended in ESC medium and re-plated onto gelatin coated flasks at a density of ~4 × 104 cells/cm2 (determined using a hemocytometer - Neubauer).All centrifugation steps with live cells were performed at 330 x g for 3 min at room temperature (RT). All ESC lines used in this study were routinely tested for mycoplasma.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Trypsinized mESCs (20 x 10e6) were washed twice in PBS. Cells were resuspended in 250 µl of PBS and fixed by the addition of an equal volume of PBS containing 2% methanol free formaldehyde (Thermo Scientific Pierce PN28906; final concentration of 1%) and incubated at RT for 10 min. Fixation was stopped by 5 min incubation with 125 mM glycine at room temperature. Fixed cells were washed in PBS and combined at this stage with 1.3x10e6 formaldehyde fixed S2 cells (Drosophila melanogaster cells; for downstream calibration of ChIP-seq data). All buffers were supplemented with 1 mM DTT and 1x Protease inhibitors (Roche, 11836170001) just prior to use. Cell pellets were resuspended in lysis buffer (50 mM Tris-HCl pH 8.1, 10 mM EDTA and 20% SDS) and incubated for 10 min at 4 °C. Lysates were diluted 1:10 in ChIP Dilution Buffer (1% Triton X-100, 2 mM EDTA, 150 mM NaCl, 2 0mM and Tris-HCl pH8.1) and sonicated, first with a single 30s pulse with a probe sonicator (labtech Soniprep 150) on ice followed by a further 45 cycles using a cooled Bioruptor (Diagenode; 1 min cycles of 30sec on / 30 sec off on ‘high’ setting at 4 °C). The sonicated extract was pre-cleared by centrifugation at 16000 g for 10 min at 4 °C. The supernatant was transferred to a fresh tube and supplemented with BSA to a final concentration of 25 mg/ml. Anti-RING1B (Cell Signalling D22F2) antibodies were pre-coupled to a protein A Dynabeads (Life Technologies; catalogue no. 10001D) at a ratio of 1 mg antibody per 30 ml of dynabead suspension by rotation at 4 °C for 1 h. 12 x 10e6 cell equivalents of lysate were added to 7.5 µg of antibody bound beads and incubated for 6 h on a rotating wheel at 4 ºC. Following incubation, bead-associated immune complexes were washed sequentially with ChIP dilution buffer, wash buffer A and wash buffer B, each for 10 min at 4 ºC on a rotating wheel followed by two washes in TE buffer at RT (wash buffer A - 1% Triton X-100, 0.1% Sodium-Deoxycolate, 0.1% SDS, 1mM EDTA, 500 mM NaCl, 50 mM HEPES pH7.9; wash buffer B – 0.5% NP40, 0.5% Sodium-Deoxycolate, 1 mM EDTA, 250 mM LiCl, and 20 mM Tris-HCl pH8.1). Chromatin was released by incubating the beads in 100 µl of elution buffer (0.1 M NaHCO3 and 1 % SDS) for 15 min at 37 ºC, followed by the addition of 50 µg of RNaseA and 6µl of 2 M Tris pH6.8 and incubation at 65 ºC for 2 h and finally by the addition of 50 µg of proteinase K and incubation at 65 ºC for 8 h to degrade proteins and reverse the cross-links. Dynabeads were removed using a magnetic rack and the chromatin purified using PCR Purification columns (Qiagen) according to manufacturer’s instructions.
Libraries were constructed using the NEBNext® Ultra™ II DNA Library Prep Kit for Illumina according to manufacturer’s instructions (NEB - catalogue no. E7645S). Library PCRs were supplemented with 2x SYBR dye (Sigma – catalogue no. S9430) so that amplification could be monitored by quantitative PCR on a Roche lightcycler 480. To allow for sample multiplexing, PCRs were performed using index primers (NEBNext Multiplex Oligos for Illumina - Set 1. Catalogue no. E7335) and amplified to linear phase. Size selection purifications following the ligation and amplification PCR steps were performed with 1x and 0.9x reaction volumes of Agencourt AMPure XP beads (Beckman Coulter - A63880). Purified libraries were combined as a 12 sample equimolar pool containing the indexes 1-12 and sequenced on an Illumina NextSeq on a single high-output flow cell (single-end 75 bp reads).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
NextSeq 550 |
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| Description |
ChIP-seq for RING1B from mESCs treated with DMSO for 24 h. Replicate 1 of 2.
ChIP-seq
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| Data processing |
Base Calling. Basecalling, quality scoring and de-multiplexing was performed directly using the NextSeq 550 software (RTAv2). Alignment. ChIP-seq data was mapped to the mouse genome (mm9 build) using bowtie2 with the --local --threads 3 -S options to generate SAM files. Processing. Using the HOMER package, SAM files were converted into tag directories and multi-mapping reads were removed using makeTagDirectory -unique -fragLength 150. Mapped regions which, due to fragment processing, extended beyond the end of the chromosomes were removed using removeOutOfBoundsReads.pl with chromosome lengths for mm9. Genome browser files (.bw) were generated using makeUCSCfile (HOMER package) with the -bigWig -fsize 1e20 -norm 10e7 options. H3K27me3 genome browser files were generated using the same parameters but normalised to a calibrator value that preserved the relative contribution of Drosophila spike-in reads between the input and immunoprecipitated samples.
Genome_build: mm9
Supplementary_files_format_and_content: .bigWig files contain information on both the genomic distribution and signal strength of the mapped reads (representative of the sample composition). .bigWig files are in an indexed binary format.
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| Submission date |
Nov 23, 2019 |
| Last update date |
Dec 17, 2019 |
| Contact name |
Rob S Illingworth |
| E-mail(s) |
robert.illingworth@ed.ac.uk
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| Phone |
01316519640
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| Organization name |
The University of Edinburgh
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| Department |
Centre for regenerative Medicine
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| Lab |
Illingworth
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| Street address |
Centre for Regenerative Medicine
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| City |
Edinburgh |
| State/province |
Midlothian |
| ZIP/Postal code |
EH16 4UU |
| Country |
United Kingdom |
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| Platform ID |
GPL21626 |
| Series (1) |
| GSE140894 |
Genome-wide profiling of transcription, H3K27me3 and RING1B occupancy in mouse ESCs following EZH1/2 inhibition |
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| Relations |
| BioSample |
SAMN13379068 |
| SRA |
SRX7205307 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4189779_RING1B_DMSO_1_10M.bigWig |
249.1 Mb |
(ftp)(http) |
BIGWIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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