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| Status |
Public on Sep 13, 2016 |
| Title |
NCCIT_H3K4me3_ChIP-Seq |
| Sample type |
SRA |
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| Source name |
NCCIT cells
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| Organism |
Homo sapiens |
| Characteristics |
cell line: NCCIT
cell type: Pluripotent embryonal carcinoma
antibody: anti-H3K4me3 (07-473, Millipore)
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| Treatment protocol |
No treatment
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| Growth protocol |
NCCIT cells were grown in RPMI (Corning) with 15% FBS. All cells were grown in 5% CO2 at 37°C to 70-80% confluence in 150 mm culture dishes.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Mononucleosomes were isolated by MNase digestion of cells, followed by formaldehyde fixing and fractionating the concentrated nucleosome mixture it on a 5-25% sucrose gradient, to purify the mononucleosome fraction away from dinucleosomes. Alternate "Quick protocol" involved controlled detergent mediated treatment of NCCIT cells and digestion with a fixed concentration of MNase that allows release of mononucleosomes into the supernatant that are crosslinked and used for ChIP. Modified Sequential ChIP involved crosslinking of the 1st Antibody to beads by DSG followed by elution with SDS/65degC and the elute used for sequential pulldown using the 2nd Antibody. DNA was extracted using Qiagen PCR purification preps from the decrosslinked eluate of sequential ChIP and was used either for western blotting or PCR.
10ng of seq ChIP DNA was amplified by WGA using Illumina kits for Library preparation. Libraries were constructed according to the protocol provided by Illumina’s TruSeq DNA Sample Preparation v2 Kit. Library prep begins with the End Repair and dA tailing followed by Illumina indexed sequencing adaptor ligation that are complementary for hybridization on the flow cell. PCR amplification was used to amplify the DNA fragments for 15 cycles and assessment of the yield and size distribution of the amplified library was performed on the Agilent 2100 Bioanalyzer using the High Sensitivity Chip. Quantitation of the libraries was performed by qPCR using the KAPA SYBER FAST qPCR Kit supplied by Kapa Biosystems according to manufactures’ protocol. Multiplex libraries were pooled to a final concentration of 2nM and cluster generation completed on Illumina’s cBot using the TruSeq SR Cluster Kit v3. Single read 50bp sequencing was performed on the Illumina HiSeq2000 with the TruSeq SBS Kit v3 (50cycles).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
AB SOLiD System 3.0 |
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| Description |
ChIP-seq with mentioned Ab
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| Data processing |
Reads were aligned to hg18 (NCBI36) using Bioscope 1.2.1, finding all alignments between the first 25 bp of the read (seed) and the reference sequence with up to two mismatches. Each match is extended to the full length of the read, scoring 1 point for matching and −2 points for mismatching bases. The read is trimmed to the length with the highest score. If there is only one alignment or if an alignment scores significantly higher than the others for the same read, it is considered unique and reported.
Samtools and Bedtools used to filterreads as below and get output as BED files: Remove duplicates and create bam file (-b) of only mapped reads (-F 4) with mapping quality (MAPQ) better than 20 (-q 20), and not more than 1 mismatches (NM is 0 or 1; NM is the number of bases that has to be edited to get a perfect match).
Peaks were called using SICER. Settings for finding H3K4me3 peaks: redundancy_threshold=1, window_size_bp=200, fragment_size=160, effective_genome_fraction=0.75, gap_size_bp=600, FDR=0.01; Settings for H3K27me3 peaks:redundancy_threshold=1, window_size_bp=200, fragment_size=160, effective_genome_fraction=0.75, gap_size_bp=1000, FDR=0.01
Genome_build: hg18
Supplementary_files_format_and_content: *.txt file; summary of all candidate islands with their statistical significance. File format/column labels: chrom, start, end, ChIP_island_read_count, CONTROL_island_read_count, p_value, fold_change, FDR_threshold
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| Submission date |
Sep 12, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
hari easwaran |
| Organization name |
Johns Hopkins University
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| Department |
Oncology
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| Lab |
Steve Baylin
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| Street address |
1650 Orleans Street, CRB1, Room 530
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| City |
BALTIMORE |
| State/province |
MD |
| ZIP/Postal code |
21209 |
| Country |
USA |
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| Platform ID |
GPL9442 |
| Series (1) |
| GSE86838 |
Genome-wide positioning of bivalent mononucleosomes |
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| Relations |
| BioSample |
SAMN05763099 |
| SRA |
SRX2160033 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2309221_JHUSB01002_002_K4_SS02.sorted.bam_FILTERED-W200-G600-islands-summary-FDR0.01.txt.gz |
1.3 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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