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| Status |
Public on Mar 31, 2017 |
| Title |
C8_Brd2KO G1E-ER4+E2 24 hr input |
| Sample type |
SRA |
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| Source name |
C8_Brd2KO G1E-ER4 cells
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| Organism |
Mus musculus |
| Characteristics |
cell type: erythroid
chip ab: none
genotype/variation: C8_Brd2KO G1E-ER4
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| Treatment protocol |
Cells were induced to differentiate (activate GATA1) by the addition of 100nM estradiol for 24 hours
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| Growth protocol |
G1E-ER4 cells were grown in IMDM+15% FBS, penicillin/streptomycin, kit ligand, monothioglycerol and Epoetin alpha in a standard tissue culture incubator at 37 degreses with 5% CO2 (as described in Weiss et al., 1997)
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
G1E-ER4 cells were crosslinked with 1% formaldehyde in PBS with gentle agitation for 10 minutes at room temperature and then quenched with glycine. Fixed cells were resuspended in 1mL Cell Lysis Buffer (10mM Tris-HCl pH 8, 10mM NaCl, 0.2% NP-40/Igepal) prepared fresh with protease inhibitors (Sigma P8340) and 1mM phenylmethylsulfonyl fluoride (PMSF) and incubated on ice for 10 minutes. Nuclei were pelleted and resuspended in 1mL Nuclear Lysis Buffer (50mM Tris-HCl pH 8, 10mM EDTA pH 8, 1% SDS, prepared fresh with protease inhibitors and PMSF), and incubated on ice for 20 minutes. Samples were then diluted with 0.6mL IP Dilution Buffer (20mM Tris-HCl pH 8, 2mM EDTA pH 8, 150mM NaCl, 1% Triton X-100, 0.01% SDS, prepared fresh with protease inhibitors and PMSF), and sonicated at 4 degrees C (Epishear, Active Motif). After sonication, samples were spun at 4 degrees C to remove debris and added to preclearing reactions containing 3.4mL IP Dilution Buffer, protein A/G agarose beads, and isotope-matched IgG. Samples were precleared for ?2 hours.
Antibody (anti-CTCF, Millipore (07-729, lot 2517762) was prebound to protein A/G agarose beads. Precleared chromatin was added to beads and IPs were performed overnight at 4 degrees C with rotation. Beads were washed once with IP Wash 1 (20mM Tris-HCl pH 8, 2mM EDTA pH 8, 50 mM NaCl, 1% Triton X-100, 0.1% SDS), twice with High Salt Buffer (20 mM Tris-HCl pH 8, 2mM EDTA pH 8, 500 mM NaCl, 1% Triton X-100, 0.01% SDS), once with IP Wash Buffer 2 (10 mM Tris-HCl pH 8, 1mM EDTA pH 8, 0.25 M LiCl, 1% NP-40/Igepal, 1% sodium deoxycholate), and twice with TE (10mM Tris-HCl pH 8, 1mM EDTA pH 8). All washes were performed on ice. Following the final wash, beads were moved to room temperature and eluted twice with 100ul of Elution Buffer (100mM NaHCO3, 1%SDS, prepared fresh) for a final eluate volume of 200ul. The following were added to each sample: 12ul of 5M NaCl, 2ul RNaseA (10mg/ml) and samples were incubated at 65C for ?1 hour. 3ul of Proteinase K (20mg/ml) was added and samples were incubated at 65C overnight.
Following overnight incubation, 10ul of 3M sodium acetate pH 5 was added to each sample and DNA was purified using the QIAquick PCR Purification kit per the manufacturer’s instructions.
Libraries were prepared according to Illumina's instructions accompanying TruSeq ChIP-seq Sample Preparation Kit. (Part# IP-202-1012). In brief, ChIP-enriched, fragmented DNA was subjected to end repair to generate blunt-end double stranded DNA, adenylation of 3’ ends, and adaptor ligation. Following ligation, SPRIselect (Beckman Coulter) beads were used at 0.9X and 0.6X for left and right side selection, respectively, to obtain an average library target size of ~300 bp. After size selection, fragments were amplified for 16 cycles, and PCR products were purified using Agencourt AMPureXP beads (Beckman Coulter).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
Basecalls using bcl2fastq-1.8.4, and parameters --no-eamss --mismatches 1
Mapping to reference genome mm9 canon with Bowtie 1.0.0 using parameters --chunkmbs 1024 -y -n 2 --best -k 1 --maxbts 800 -l 28 -e 80 --sam-nohead --sam
Wiggle and peaks called using MACS with parameters --format BAM --gsize 1870000000 --tsize 36 --bw 120 --mfold 12 --wig --space 1
Filter blacklist regions from peaks, and convert the *peaks.xls file from MACs to broadpeak format (see UCSC Genome Browser for format specs)
Genome_build: mm9
Supplementary_files_format_and_content: bigWig file with read coverage, peaks in broadpeak format
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| Submission date |
Apr 21, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Gerd A Blobel |
| E-mail(s) |
blobel@email.chop.edu
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| Organization name |
University of Pennsylvania Children's Hospital of Philadelphia
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| Department |
Pediatrics/Hematology
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| Street address |
3615 Civic Center Blvd, ARC 315
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| City |
Philadelphia |
| State/province |
PA |
| ZIP/Postal code |
19104 |
| Country |
USA |
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| Platform ID |
GPL19057 |
| Series (2) |
| GSE80527 |
The BET protein BRD2 cooperates with CTCF to enforce a transcriptional boundary |
| GSE95804 |
The BET protein BRD2 cooperates with CTCF to enforce transcriptional and architectural boundaries |
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| Relations |
| BioSample |
SAMN04883343 |
| SRA |
SRX1718164 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2129860_868.bigWig |
2.9 Gb |
(ftp)(http) |
BIGWIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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