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Sample GSM1059177 Query DataSets for GSM1059177
Status Public on Dec 06, 2013
Title wdr5 chipseq rep2
Sample type SRA
Source name wdr5 ChIP-seq
Organism Mus musculus
Characteristics strain background: C57BL/6
cell type origin: embryonic stem cells
cell type: ES-derived neural progenitor cells
chip antibody: Wdr5 antibody
chip antibody vendor: Bethyl
chip antibody cat. #: A302-429A
chip antibody lot #: A302-429A-1
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).
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.
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.
Library strategy ChIP-Seq
Library source genomic
Library selection ChIP
Instrument model Illumina MiSeq
Description Sample 7
Data processing Basecalls performed using CASAVA version 1.6
Data quality was assessed with 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:, and each line contains the fields: chrom, chromstart, chromend.
Submission date Jan 02, 2013
Last update date May 15, 2019
Contact name Stephen D Ayers
Phone 713-441-8693
Fax 713-793-7162
Organization name Methodist Hospital Research Institute
Department Genomic Medicine
Lab Genomic Medicine
Street address 6565 Fannin Street F8-060
City Houston
State/province Texas
ZIP/Postal code 77030
Country USA
Platform ID GPL16417
Series (1)
GSE43247 ChIP-seq analysis of PHF20 and Wdr5 binding sites in ESCs
SRA SRX213763
BioSample SAMN01881852

Supplementary file Size Download File type/resource
GSM1059177_MISEQ7.bed.gz 27.1 Mb (ftp)(http) BED
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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