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| Status |
Public on Jan 21, 2016 |
| Title |
H2AZ_WT_ESC_Rep1 |
| Sample type |
SRA |
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| Source name |
ES cells
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| Organism |
Mus musculus |
| Characteristics |
strain: V6.5 (129SvJaexC57BL/6)
cell type: Embryonic stem cells
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| 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).
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| 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.
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted using TRIzol. Strand specific libraries were prepared with the purified RNA subjected to oligo (dT) selection, fragmentation and first and double strand synthesis with the Illumina Tru-Seq kit using manufacturer’s instructions. DNA fragments above 30bp was purified using SPRI-TE beads according to manufacturer’s instructions. The purified DNA was end repaired and single A bases were added for adaptor ligations. The adaptor-ligated DNA was then subjected to Pippin purification to select for 300bp DNA fragments. These fragments were enriched and barcoded by PCR for multiplexing. A final Pippin purification was performed to clean up the barcoded RNA-Seq libraries.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
Illumina Offline BaseCaller1.9.3 software used for basecalling.
The reads were mapped to mm9 genome using OLego (Wu et al.,2013) (1.1.2). Known junctions extracted from igenome (Illumina) were provided to OLego to maximize the sensitivity. Default options were used for OLego. Afterwards, HTSeq-count (0.5.4p1) was used to summarize the read counts mapped to each transcript (http://www-huber.embl.de/users/anders/HTSeq/doc/overview.html).
The RPKM values were computed by normalizing the raw read counts with the transcript lengths and total numbers of reads for each sample. Raw read counts were used to detect differentially expressed genes using DEseq (1.10.1) with default parameters (Anders et al., 2010).
Genome_build: mm9
Supplementary_files_format_and_content: A tab-delimited text file that includes RPKM values for each Sample, and log2(fold-changes) among samples at each time-point.
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| Submission date |
Dec 11, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Laurie A Boyer |
| E-mail(s) |
lboyer@mit.edu
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| Phone |
617 324-3335
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| Organization name |
Massachusetts Institute of Technology
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| Department |
Biology
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| Lab |
Boyer
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| Street address |
77 Massachusetts Avenue
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| City |
Cambridge |
| State/province |
MA |
| ZIP/Postal code |
02139 |
| Country |
USA |
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| Platform ID |
GPL13112 |
| Series (2) |
| GSE53207 |
H2A.Z.1 mono-ubiquitylation antagonizes BRD2 to maintain poised chromatin in ESCs [RNA-seq] |
| GSE53208 |
H2A.Z.1 mono-ubiquitylation antagonizes BRD2 to maintain poised chromatin in ESCs |
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| Relations |
| BioSample |
SAMN02440135 |
| SRA |
SRX390113 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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