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| Status |
Public on Jan 28, 2018 |
| Title |
BRD4-IPS-AmacFibr_F_N-fG140.02 |
| Sample type |
SRA |
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| Source name |
Cell Line
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| Organism |
Mus musculus |
| Characteristics |
strain background: C57BL/6
cell line: AmacFibr_F_N-fG140.02
cell type: iPSCs from fibroblast Amacrine cell CanNotMakeRetina fG140.02
chip antibody: BRD4 (Bethyl,A301-985A)
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
After removal of IRMEFs, iPS cells were collected after washing with PBS. Then cells were subjected to crosslinking in 1% formaldehyde (Thermo scientific, 28906) for 10 minutes at room temperature, followed by the addition of glycine at the final concentration of 0.125 M to quench the reaction. After centrifuge, cells were washed with cold PBS, counted and pelleted as 10 million cells for each nuclei preparation.
The nuclei were isolated and prepared for shearing using TruChIP chromatin shearing kit (Covaris, 520127) according to manufacturer’s protocol. After shearing, ChIP was performed using the iDeal ChIP-seq kit (Diagenode, C01010051). The antibodies used were: anti-H3K4me3 (Diagenode, C15410003-50), anti-H3K4me2 (Abcam, ab7766), anti-H3K4me1 (Abcam, ab8895), anti-H3K9/14ac (Diagenode, C15410200), anti-H3K27ac (Abcam, ab4729), anti-H3K36me3 (Active Motif, 61101), anti-H3K27me3 (Active Motif, 39155), and anti-CTCF (Active Motif, 61311). After de-crosslinking, DNA was extracted using MinElute PCR-purification kit (Qiagen, 28006) or Agencourt AMPure XP beads (Beckman Coulter, A63881) and subjected to library preparation using NEBNext ChIP-Seq Library Prep Master Mix Set for illumina (NEB, E6240L). The ChIP experiments and library preparation for antibodies including anti-RNA polymerase II (Abcam, ab5095), anti-Brd4 (Bethyl Laboratories, A301-985A) and anti-H3K9me3 (Active Motif, 39161), were performed by Active Motif.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
Basecalls performed using CASAVA
BWA (version 0.5.9-r26-dev, default parameter) to align the ChIP-Seq reads to mouse genome mm9(MGSCv37 from Sanger), Picard(version 1.65(1160)) then have been used for marking duplicated reads. Then only non-duplicated reads with have been kept by samtools (parameter “-q 1 -F 1024” version 0.1.18 (r982:295)). We followed the ENCODE criterion to quality control (QC) the data that non-duplicated version of SPP (version 1.11) have been used to draw cross-correlation and calculated relative strand correlation value (RSC) under support of R (version 2.14.0) with packages caTools(version 1.17) and bitops(version 1.0-6) and estimated the fragment size.
We required > 10M unique mapped reads for point-source factor (H3K4me2/3, H3K9/14Ac, H3K27Ac, CTCF, RNAPolII, BRD4) and RSC > 1. We required 20M unique mapped reads for broad markers (H3K9me3, H3K27me3, H3K36me3). We required 10M unique mapped reads for INPUTs and RSC < 1. We noticed H3K4me1 is point-source factor in some stages while broad in other stages so we QC H3K4me1 as broad markers. Then upon manually inspection, the cross-correlation plot generated by SPP, the best fragment size estimated (the smallest fragment size estimated by SPP in all our cases) were used to extend each reads and generate bigwig file to view on IGV (version 2.3.40).
All profiles were manually inspected for clear peaks and good signal to noise separation. We followed ENCODE guideline for quality control.
We extended reads and generate the final bigwig track normalized to 30M reads for visualization.
Genome_build: mm9(MGSCv37)
Supplementary_files_format_and_content: bigwig
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| Submission date |
Jan 26, 2018 |
| Last update date |
Jan 29, 2018 |
| Contact name |
Beisi Xu |
| E-mail(s) |
beisi.xu@stjude.org
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| Organization name |
St Jude Children's Research Hosipital
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| Department |
Center for Applied Bioinformatics
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| Street address |
262 Danny Thomas Pl
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| City |
Memphis |
| State/province |
Tennessee |
| ZIP/Postal code |
38105 |
| Country |
USA |
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| Platform ID |
GPL13112 |
| Series (2) |
| GSE87064 |
The Dynamic Epigenetic Landscape of the Retina During Development, Reprogramming, and Tumorigenesis |
| GSE109712 |
Retinal Cell Type Epigenetic Memory Predicts Reprogramming Efficiency and Retinogenesis in 3D Organoid Cultures [ChIP-seq_Mm] |
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| Relations |
| BioSample |
SAMN08405591 |
| SRA |
SRX3602877 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2948951_BRD4-IPS-AmacFibr_F_N-fG140.02.bw |
224.1 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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