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Sample GSM1287699 Query DataSets for GSM1287699
Status Public on Jan 21, 2016
Title H2AZ_WT ChIP-Seq
Sample type SRA
Source name ES cells
Organism Mus musculus
Characteristics strain: V6.5 (129SvJaexC57BL/6)
cell type: Embryonic stem cells
chip antibody: Abcam:Ab290
Treatment protocol H2AZ-YFP, and H2A.Z.K3R3-YFP constructs were modified from vectors generated in Sarcinella et al., 2007. The H2AZ.K3R3 mutant is a triple point mutant in the C-terminus of H2AZ with replacement of K119,K120, K123 with arginines, with the GFP in this vector being replaced by YFP, such that it is in frame C-terminal to the cDNA. EcoRI and XbaI digestion was performed to place H2AZ, AP3 and H2A cDNA in frame. This vector contains a CMV-promoter driven by an rtTA drug inducible system. The resulting lentiviral constructs were transfected into 293 cells using the protocol outlined by the RNAi consortium (BROAD Institute, The viral supernatant generated 48hrs after transfection was used to infect KH2 ESCs (Beard, 2006) to generate wildtype and mutant H2AZ transgenic ESC lines. The YFP transgenic ESCs were induced with 1µg/ml of doxycycline and FACS sorted for YFP positive cells. In order to study the selective impact of the H2AZ mutants, lentiviral constructs expressing short hairpins specifically directed at the 3’ UTR of endogenous H2AZ were introduced into the wild-type and mutant H2AZ transgenic KH2 ESC lines using RNA. Sequences of the different H2AZ 3’UTR-directed hairpin oligos are as follows: sh#2 5’- AACAGCTGTCCAGTGTTGGTG-3’; sh#5 5’- AATTAGCCTTCCAACCAACCA-3’. Hairpin oligos were annealed and cloned into pLKO.1 vector (Sigma) as detailed by the RNAi consortium, BROAD ( Blasticidin was used as a selection marker for the generation of endogenous H2AZ-depleted transgenic KH2 ESCs. The puromycin marker in the pLKO.1 vector was removed by digestion with BamHI and KpnI and replaced with blasticidin. The blasticidin cDNA was PCR amplified from pLenti6.2/V5-DEST Gateway® Vector (Invitrogen). V6.5 (129SvJae and C57BL/6) and the YFP transgenic ESCs were cultured as previously described (Boyer et al., 2006). The endogenous H2AZ-depleted transgenic KH2 ESCs were similarly cultured with the addition of blasticidin (5µg/ml) on blasticidin-resistant feeder cells (Iuchi, 2006).
Growth protocol V6.5 (129SvJae and C57BL/6; male) ESCs were plated with irradiated murine embryonic fibroblasts (MEFs) and grown under typical ESC conditions on gelatinized tissue culture plates. Briefly, cells were grown in Knockout DMEM supplemented with 10% fetal bovine serum, leukemia inhibitory factor (LIF), non-essential amino acids, L-glutamine, and penicillin/streptomycin as previously described (Boyer et al., 2006). ESCs used for the majority experiments, including ChIP-Seq, RNA-Seq and RT-qPCR, were plated without irradiated MEFs for the final passage.
Extracted molecule genomic DNA
Extraction protocol The ChIP protocol has been adapted from previous studies (Lee et al., 2006; Boyer et al., 2006, Creyghton et al., 2008) with the following modification. Diagenode Bioruptor was used to sonicate with 30 cycles of 30 sec on, 30 sec off. The samples were sonicated in 15ml polystyrene tubes at 4°C while samples were immersed in ice cold water. Antibodies used for ChIP include: ~200 ng of DNA was submitted to SPRI-works Fragment Library System I (Beckman Coulter) for each library prepared. Briefly, the DNA is subjected to size selection (200-400bp) with magnetic beads, end repaired, then a single adenine nucleotide is added to allow for directional ligation of adaptors. For this study, a 1:100 dilution of Single End read adapters (Illumina) was used in the ligation reaction. Each sample was then amplified for 19 cycles, using the Illumina protocol, adding additional bases from the PCR primers that aid sequences to anneal to the Illumina Genome Analyzer flow cell. The samples were then purified on a Qiagen MinElute column, and libraries were quantified by Quant-it DNA Assay (Invitrogen, Q-33120), and examined for proper size and structure by Bioanalyzer (Agilent) and qPCR.
Library strategy ChIP-Seq
Library source genomic
Library selection ChIP
Instrument model Illumina Genome Analyzer IIx
Data processing Illumina Offline BaseCaller1.9.3 software used for basecalling.
ChIP-seq reads were aligned to the mm9 genome assembly using Bowtie 0.12.3, with the following parameters --solexa1.3-quals --best --strata -m 1 -n 2 -p 8 -S.
Sequences were extended +200 bp for histone marks and allocated in 25-bp bins.
A Poissonian model was used to determine statistically enriched bins with a P-value threshold set at 1x10-9 as described previously (Marson et al., 2008).  Additionally, we required that genomic bins were at least 5 fold over input to be considered enriched peaks. 
Genome_build: mm9
Supplementary_files_format_and_content: wig files were generated using an in-house perl script interacting with Bedtools (as described in Wamstadt et al. Cell 2012).
Submission date Dec 11, 2013
Last update date May 15, 2019
Contact name Laurie A Boyer
Phone 617 324-3335
Organization name Massachusetts Institute of Technology
Department Biology
Lab Boyer
Street address 77 Massachusetts Avenue
City Cambridge
State/province MA
ZIP/Postal code 02139
Country USA
Platform ID GPL11002
Series (2)
GSE53206 H2A.Z.1 mono-ubiquitylation antagonizes BRD2 to maintain poised chromatin in ESCs [ChIP-seq]
GSE53208 H2A.Z.1 mono-ubiquitylation antagonizes BRD2 to maintain poised chromatin in ESCs
BioSample SAMN02440136
SRA SRX390111

Supplementary file Size Download File type/resource
GSM1287699_H2AZ_WT.1e-09.bed.gz 677.7 Kb (ftp)(http) BED
GSM1287699_H2AZ_WT.1e-12.bed.gz 658.4 Kb (ftp)(http) BED
GSM1287699_H2AZ_WT.WIG.gz 11.0 Mb (ftp)(http) WIG
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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