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| Status |
Public on Oct 17, 2013 |
| Title |
KLF3, immunoprecipitation |
| Sample type |
SRA |
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| Source name |
Klf3-/- murine embryonic fibroblasts, rescued with Klf3-V5
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| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
cell type: MEFs
genotype/variation: Klf3-/- rescued with Klf3-V5
antibody: anti-V5 antibody (Cat# R960-CUS, Life Technologies, Carlsbad, CA)
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| Growth protocol |
Cells were grown in DMEM supplemented with 10% FCS and with 1X PSG
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
ChIP was conducted in duplicate on Klf3-/- MEFs expressing recombinant Klf3, ΔDL or DBD. Aproximately 5x107 cells were used for each experiment and ChIP was conducted as previously described (Schmidt et al. 2009) using an anti-V5 antibody (Cat# R960-CUS, Life Technologies, Carlsbad, CA).
Library preparation was performed using the TruSeq DNA Sample Preparation Kit (Cat# FC-121-2001, Illumina, San Diego, CA) according to manufacturer’s instructions with minor modifications. Adapter sequences were diluted 1/40 before use and following adapter ligation, the library size extracted from the gel was 100-280 bp (excluding adapters) in line with the size of sonicated fragments.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
rep1 and 2
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| Data processing |
Libraries (6 inputs and 6 IP samples) were multiplexed into four lanes using sample specific adapters such that there were 3 samples per lane. Samples were sequenced using 50 bp chemistry on the HiSeq 2000 (Illumina, San Diego, CA). Quality control on the sequence data was performed using FastQC v0.10.1 available from http://www.bioinformatics.babraham.ac.uk/projects/fastqc/.
Base-calling and demultiplexing was performed using the standard Illumina software packaged with the HiSeq2000. Quality control on the sequence data was performed using FastQC v0.10.1 available from http://www.bioinformatics.babraham.ac.uk/projects/fastqc/.
Reads were aligned to the mm9/NCBI37 Mus musculus genome using Bowtie2 v2.0.0-beta7 (Langmead and Salzberg 2012). In the first round, Bowtie2 was set to --very-sensitive and –D 40. Non-aligned reads were subjected to a second round of alignment where the read could be soft clipped by running Bowtie2 with the switch --very-sensitive-local. Resulting alignments were sorted, merged and indexed using Samtools v0.1.18 (Li et al. 2009).
Peak calling and downstream analysis was primarily performed using the HOMER software package v4.1 (available from http://biowhat.ucsd.edu/homer/ngs/index.html) (Heinz et al. 2010). The script findPeaks.pl was used to for peak discovery using the paired input sample as a control with the settings -style factor, -F 5 and -L 5, requiring 5x fold enrichment over input and 5x fold enrichment over background (surrounding 10 kb) to call a peak. Peaks were subjected to an FDR cut-off of 0.001. Peaks were merged using mergePeaks using the switch -d given meaning that peaks had to literally overlap in genomic space to be considered overlapping. Peak lists were annotated using annotatePeaks.pl using the HOMER annotation set for mm9/NCBI37.
HOMER was used to quantify ChIP tag density at peak locations across the genome. Tags were counted within 400 bp around the peak centre (as peak widths could vary across the three different samples). All tag counts were normalised to 100M reads and were thus expressed as reads/100M reads to allow comparison across samples. HOMER was also used to create bedgraph files using the makeUCSCfile program.
Genome_build: mm9/NCBI37
Supplementary_files_format_and_content: Processed data files include 1) a normalised ChIP tag density track (.bedgraph). This tag density track reflects the normalised tag density across the merged replicates. 2) Peak lists for each individual replicate (tab-delimited-txt). 3) Peak lists that reflect the overlap between replicates and include log2 normalised tag counts for each peak (tab-delimited-txt). 4) A matrix table that includes peaks across all three conditions and log2 normalised tag counts for each peak (tab-delimited-txt).
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| Submission date |
Feb 28, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Jon Burdach |
| Organization name |
University of New South Wales
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| Department |
BABS
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| Street address |
UNSW
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| City |
Sydney |
| State/province |
NSW |
| ZIP/Postal code |
2052 |
| Country |
Australia |
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| Platform ID |
GPL13112 |
| Series (1) |
| GSE44748 |
Genome-wide binding profiles of KLF3 and KLF3 mutants in MEF cells |
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| Relations |
| SRA |
SRX248398 |
| BioSample |
SAMN01974697 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1090065_KLF3-IP.bedgraph.gz |
224.1 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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