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| Status |
Public on May 20, 2014 |
| Title |
LS174T, GATA6 ChIP-seq 2 |
| Sample type |
SRA |
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| Source name |
LS174T, GATA6 ChIP
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| Organism |
Homo sapiens |
| Characteristics |
cell line: LS174T
cell type: colorectal carcinoma
antibody: anti-hGATA6 (R&D AF1700)
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| Growth protocol |
LS174T cells were grown in DMEM plus 10% FBS. When cells were at 70-80% confluence, proteins were cross-linked to DNA with formaldehyde 1% 15 minutes.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were lysed in 0.5% SDS, lysates were sonicated and clarified. Chromatin was diluted 5 fold and incubated with beads for pre-clearing. After pre-clearing, chromatin was incubated o/n with anti-GATA6, anti-H3K4me3, anti-H3K4me1, or anti-H3K27ac antibodies.
20ng of DNA, as quantitated by fluorometry, was resolved by electrophoresis and fractions of 50-250bp were extracted. Fractions were processed through subsequent enzymatic treatments of end-repair, dA-tailing, and ligation to adapters as in Illumina's "TruSeq DNA Sample Preparation Guide" (part # 15005180 Rev. C). Adapter-ligated libraries were completed by limited-cycle PCR with Illumina PE primers (12 cycles). The resulting purified DNA library was applied to an Illumina flow cell for cluster generation (TruSeq cluster generation kit v5) and sequenced on the Genome Analyzer IIx with SBS TruSeq v5 reagents by following manufacturer's protocols.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer IIx |
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| Data processing |
Image analysis and per-cycle basecalling was performed with Illumina Real Time Analysis software (RTA1.13). Conversion to FASTQ read format and sequence alignment with the ELAND algorithm (v2e) was performed with CASAVA-1.8 (Illumina).
ChIP-seq reads were aligned to the hg19 genome assembly using BWA version 0.5.9-r16 allowing 0-1 mismatches
Unique aligned reads were converted to BED format
GATA6 ChIP-seq replicates & Input DNA aligment files were normalized to the same number of reads randomly.
Peaks were called using macs version 2.0.9 with the following setting: format (BED), effective genome size (2.70e+09), band width (300), model fold (10,30), qvalue cutoff (5.00e-02)
Reproducibility of replicates was assessed ranking the peaks of each by q-value. For each replicate, top 40% of peaks were evaluated to be present in the list of identified peaks for the other replicates with the method described by Chikina et al. (Chikina, MD. And Troyanskaya, OG. Ann effective statistical evaluation of ChIPseq dataset similarity. Bioinformatics 28, 607-613. (2012))
Such as for all replicates more of the 85% of top 40% significant peaks were present in the others and the number of peaks identified for each replicate don't differ by more than a factor of 2, reads from replicates were combined to repeat the peak finding analysis as described above, significant peaks obtained following this strategy were used for further analysis
The reads after merge the replicates of the ChIP-seq and normalize with the input were directionally extended to 300 bp, and for each base pair in the genome the number of overlapping sequence reads was determined and averaged over a 10 bp window to create a wig file to visualize the data in the University of California Santa Cruz genome browser
Genome_build: GRCh37
Supplementary_files_format_and_content: peak file was generated with macs version 2.0.9
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| Submission date |
Jul 29, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Camille Stephan Otto Attolini |
| Organization name |
IRB Barcelona
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| Department |
Biostatistics and bioinformatics Unit
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| Street address |
Baldiri Reixac 10
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| City |
Barcelona |
| State/province |
Select a State or Province |
| ZIP/Postal code |
08028 |
| Country |
Spain |
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| Platform ID |
GPL10999 |
| Series (2) |
| GSE49320 |
Genome-wide map of GATA6 DNA binding in human CRC cells |
| GSE56897 |
The transcription factor GATA6 allows self-renewal of colon adenoma stem cells by repressing BMP gene expression |
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| Relations |
| BioSample |
SAMN02296805 |
| SRA |
SRX328778 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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