 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Jan 21, 2016 |
| Title |
H2AZ_K3R3 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, http://www.broadinstitute.org/rnai/public/). 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 (http://www.broadinstitute.org/rnai/trc/lib). 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 |
| E-mail(s) |
lboyer@mit.edu
|
| 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 |
|
| Relations |
| BioSample |
SAMN02440138 |
| SRA |
SRX390112 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1287700_H2AZ_K3R3.1e-09.bed.gz |
769.4 Kb |
(ftp)(http) |
BED |
| GSM1287700_H2AZ_K3R3.1e-12.bed.gz |
742.8 Kb |
(ftp)(http) |
BED |
| GSM1287700_H2AZ_K3R3.WIG.gz |
12.0 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
