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| Status |
Public on Jul 01, 2015 |
| Title |
WT-MEF2B-V5-ChIP-rep2 |
| Sample type |
SRA |
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| Source name |
WT MEF2B-V5 cells
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| Organism |
Homo sapiens |
| Characteristics |
cell line: HEK293A
chip antibody: V5 (Invitrogen, R960-25)
expression: WT-MEF2B-V5
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| Growth protocol |
Cells were cultured in DMEM with 10% FBS
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Lysates were clarified from sonicated nuclei and MEF2B-DNA complexes were isolated with antibody.
Library construction was carried out on the Bravo liquid handling platform using VWorks Automation Control Software (Agilent Automation). Samples were first subjected to end repair using T4 Polynucleotide Kinase (NEB), T4 DNA Polymerase (NEB) and Klenow DNA Polymerase (NEB) at room temperature for half an hour. DNA was purified using PEG-Sera Mag Speedbeads (Fisher), with 13.87% final PEG concentration. A-tailing was then performed using Klenow exo minus (NEB) at 37oC for 30 minutes. Products were purified as before. Illumina short sequencing adaptors were ligated to A-tailed product using T4 DNA Ligase (NEB) at room temperature overnight. To remove adaptor dimers and library fragments below 200 bp, products were purified twice using PEG-Sera Mag Speedbeads with 8.89% and 10.91% final PEG concentrations. Adaptor ligated libraries were PCR amplified and barcoded using custom indexing primers, Illumina PCR primer 1.0 and 0.5 U of Phusion Hot Start II (Fisher). The initial denaturation step at 98oC for 30 seconds was followed by 13 cycles of 15 seconds at 98oC, 30 seconds at 65oC and 30 seconds at 72oC, and a final step at 72oC for 5 minutes. Amplified libraries were purified using PEG-Sera Mag Speedbeads with 9.19% final PEG concentration. Libraries were quantified using a Qubit HS DNA assay (Invitrogen) and equal-molar amounts were pooled. Each pool was quantified for sequencing with Kapa SYBR Fast Complete Universal qPCR kit (Kapa Biosystems). ChIP libraries were sequenced on the Illumina HiSeq 2000/2500 platform using v3 chemistry and HiSeq Control Software version 2.0.10. MEF2B-V5 ChIP libraries were sequenced 8 per lane. Input DNA was sequenced 14 per lane.
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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 |
Sequencing data were aligned to GRCh37-lite genome-plus-junctions reference (http://www.bcgsc.ca/downloads/genomes/9606/hg19/1000genomes/bwa_ind/genome) using BWA (version 0.5.7). BWA "aln" and "sampe" were run with default parameters. Reads failing the Illumina chastity filter were flagged with a custom script, and duplicated reads were flagged with Picard Tools (version 1.31, http://broadinstitute.github.io/picard/). Unmapped reads, optical duplicates and PCR duplicates were removed.
Wig files were produced by BAM2WIG (http://www.epigenomes.ca/tools.html).
For assessment of data quality peaks were identified at a false discovery rate of 0.01 using FindPeaks version FP4.0.15
To identify peaks present in ChIP samples that were not present in sequenced input DNA (false discovery rate of 0.05) we used MACS2 version 2.0.10.20131010
Intersects between peak lists were obtained using ChIPseek. Genes associated with peaks were identified using GREAT
Genome_build: hg19
Supplementary_files_format_and_content: NarrowPeak files were produced using MACS2 to compare ChIP samples to input control samples. Peak files were produced using FindPeaks version FP4.0.15 for quality control purposes.
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| Submission date |
Mar 31, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Patrick Plettner |
| E-mail(s) |
pplettner@bcgsc.ca
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| Organization name |
BC Cancer Agency GSC
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| Street address |
100-570 West 7th Avenue
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| City |
Vancouver |
| State/province |
BC |
| ZIP/Postal code |
V5Z 4S6 |
| Country |
Canada |
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| Platform ID |
GPL11154 |
| Series (2) |
| GSE67450 |
The MEF2B Regulatory Network - MEF2B-V5 ChIP-seq data |
| GSE67458 |
The MEF2B Regulatory Network |
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| Relations |
| BioSample |
SAMN03453091 |
| SRA |
SRX974291 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1647185_C3TRLACXX_1_ACAGTG_FDR_mc_triangle_standard.peaks.txt.gz |
23.2 Mb |
(ftp)(http) |
TXT |
| GSM1647185_C3TRLACXX_1_ACAGTG_peaks.narrowPeak.gz |
449.3 Kb |
(ftp)(http) |
NARROWPEAK |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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