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| Status |
Public on Dec 06, 2013 |
| Title |
polII chipseq rep1 |
| Sample type |
SRA |
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| Source name |
polII ChIP-seq
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| Organism |
Mus musculus |
| Characteristics |
strain background: C57BL/6
cell type origin: embryonic stem cells
cell type: ES-derived neural progenitor cells
chip antibody: Polymerase II antibody
chip antibody vendor: Abcam
chip antibody cat. #: ab5408
chip antibody lot #: gr258-5
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| Treatment protocol |
ChIP assay was performed according to the Imprint Ultra Chromatin Immunoprecipitation Kit manual (Sigma), Briefly, cells were grown to an approximate final count of 1-5×107 cells for each reaction. Cells were cross-linked with 1% formaldehyde solution for 10 min at room temperature with gentle agitation and quenched with 0.125M glycine. Cells were rinsed twice with 1×PBS, flash frozen and stored at –80°C. Cells were resuspended, lysed, and sonicated to solubilize and shear crosslinked DNA. The resulting chromatin extract was incubated overnight at 4°C with 10 mg antibody. Next day, each sample was added 15 ml blocked beads and then incubated at 4°C for 1 h. Beads were washed 5 times with RIPA buffer, once with TE containing 50 mM NaCl and complexes were eluted from beads in elution buffer by heating at 65°C. Reverse crosslinking was performed overnight at 65°C. Input DNA (reserved from sonication) was concurrently treated for crosslink reversal. DNA were treated with RNaseA, proteinase K and purified. Primary antibodies used for IP were: PHF20 (PHF20 antibody Cell signaling Cat# 3934S lot#1), Wdr5 (Wdr5 antibody Bethyl Cat# A302-429A lot# A302-429A-1), mouse/rabbit IgG and RNA Polymerase II (Polymerase II antibody abcam cat#ab5408 lot#gr258-5). Relative fold enrichments were calculated by determining the immunoprecipitation efficiency (ratios of the amount of immunoprecipitated DNA to that of the input sample).
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| Growth protocol |
mESCs and miPSCs were cultured in mESC medium (DMEM with 15% FBS, 1 mM L-glutamine (Invitrogen), 1% nonessential amino acids (Invitrogen), 0.1 mM β-mercaptoethanol (Sigma) and 1,000 U ml−1 LIF (Santa cruz)) on irradiated feeder cells.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Clarified lysates were prepared from sonicated nuclei and histone-DNA complexes were isolated according to manufacturer's standard protocols, with antibodies specific for PHF20 (Cell signaling Cat# 3934S lot#1), Wdr5 (Bethyl Cat# A302-429A lot# A302-429A-1) or PolII (abcam cat#ab5408 lot#gr258-5).
Libraries were prepared with Illumina's DNA ChIP-Seq Sample Kit (Part# 0801-0303), according to manufacturer's standard protocols. Briefly, DNA was end-repaired using T4 DNA polymerase, E. Coli DNA Pol I large fragment and T4 polynucleotide kinase. Blunt, phosphorylated ends were treated with Klenow fragment (32 to 52 exo minus) and dATP to yield a protruding 3- 'A' base (A-Tailing) for ligation of Illumina's adapters. After adapter ligation, DNA was PCR-amplified with Illumina primers for 15 cycles and library fragments of ~250 bp purified. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on an Illumina MiSeq following manufacturer's protocols.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina MiSeq |
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| Description |
Sample 4
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| Data processing |
Basecalls performed using CASAVA version 1.6
Data quality was assessed with fastqc (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/)
ChIP-seq reads were aligned to the mm9 genome assembly using bowtie 0.12.7 using --trim5 2 --trim3 5
Peaks of enriched binding were generated using QuEST version 2.4.
Genome_build: mm9
Supplementary_files_format_and_content: Standard bed format, containing reads mapped by bowtie. Lines adhere to the specification on: http://genome.ucsc.edu/FAQ/FAQformat.html#format1, and each line contains the fields: chrom, chromstart, chromend.
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| Submission date |
Jan 02, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Stephen D Ayers |
| E-mail(s) |
sda6@cornell.edu
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| Phone |
713-441-8693
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| Fax |
713-793-7162
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| URL |
http://www.methodisthealth.com/stephen-ayers-phd
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| Organization name |
Methodist Hospital Research Institute
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| Department |
Genomic Medicine
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| Lab |
Genomic Medicine
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| Street address |
6565 Fannin Street F8-060
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| City |
Houston |
| State/province |
Texas |
| ZIP/Postal code |
77030 |
| Country |
USA |
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| Platform ID |
GPL16417 |
| Series (1) |
| GSE43247 |
ChIP-seq analysis of PHF20 and Wdr5 binding sites in ESCs |
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| Relations |
| SRA |
SRX213760 |
| BioSample |
SAMN01881849 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1059174_MISEQ4.bed.gz |
30.2 Mb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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