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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Dec 20, 2012 |
| Title |
mES_Mnase_shluc_ctrl_for_H2AZKD |
| Sample type |
SRA |
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| Source name |
mouse ES cells, shluc control, MNase
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| Organism |
Mus musculus |
| Characteristics |
cell line: CMTi-1
cell type: embryonic stem cells
condition: shluc control for shH2A.Z mES cells
antibody (vendor: catalog#, or reference): none
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| Treatment protocol |
Lentiviral Infections: The RNA interference construct targeting mouse H2A.Z was generated by inserting the cDNA sequence of H2A.Z (or MLL4) into pGreenPuro Lentivector (containing a GFP marker) as described in the manual (System Biosciences). The lentiviral particles were packaged by transfecting 293T cells with the shRNA constructs or shLuciferase constructs for 2 days, and lentiviral supernatants were then harvested. Mouse ES cells were transduced with the lentiviral supernatants. After 4 days, 10-50 GFP+ cells were sorted and plated into a feeder coated 6-well plate. One week later, individual colonies were selected, expanded, and knockdown efficiency was analyzed by RT-PCR and Western blot.
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| Growth protocol |
CMTi-1 and R1 murine ES cell lines were routinely cultured on feeder-coated dishes in ES-qualified DMEM (Cat #SLM-220-B; Millipore) supplemented with 20% ES-qualified FES (Cat #SCRR-30-2020; ATCC), 2mM L-glutamine (Cat #TMS-002-C; Millipore), 100 units/ml penicillin/streptomycin (Cat #TMS-AB2-C; Millipore), 0.1mM non-essential amino acids ( Cat # TMS-001-C; Millipore), 0.1mM2-mercaptoethanol (Cat #ES-007-E; Millipore), 1mM sodium pyruvate (Cat #25-000-CI; Cellgro), and 1,000 units/ml LIF (Esgro, Cat #ESG1107; Millipore). The cells were trypsinized and replated or re-fed every second day. To generate EBs, ES cells were dissociated into a suspension of single cells using trypsin and then diluted to 10,000 cells/ml in 6-well petri dishs (Corning) in ES medium (see growth conditions for ES cells) lacking leukemia inhibitory factor (LIF). Cultures were maintained in a humidified chamber under 5% CO2 at 37C. EBs were allowed to grow for up to 14 days.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Library constructions for BNase-Seq, ChIP-Seq, MNase-Seq and RNA-Seq follow the protocols as described previously in (Epigenetics Chromatin. 2012 5:10. PMID: 22734930), (Cell. 2007 May 18;129(4):823-37. PMID: 17512414), (Cell. 2008 Mar 7;132(5):887-98. PMID: 18329373) and (Nucleic Acids Res. 2009 Sep;37(16):e106. PMID: 19528076), respectively.
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| Library strategy |
MNase-Seq |
| Library source |
genomic |
| Library selection |
MNase |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
Direct sequencing following MNase digestion on x-linked chromatin.
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| Data processing |
GA2804-CMT1-shLuc-a-MNase-0.1U_r520l1.bed.gz; genome build: mm8
Sequence reads were obtained and mapped to the mouse (mm8) genome with Bowtie. Uniquely mapped reads were kept for each lane, and for positions where multiple reads were mapped, only one read was retained (except for RNA-Seq libraries).
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| Submission date |
Dec 15, 2011 |
| Last update date |
May 15, 2019 |
| Contact name |
Gangqing Hu |
| E-mail(s) |
michael.hu@hsc.wvu.edu
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| Organization name |
West Virginia University
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| Department |
MicroBiology, Immunology, and Cell Biology
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| Lab |
2072A, HSC North, Floor 2
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| Street address |
64 Medical Center Drive
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| City |
Morgantown |
| State/province |
West Virginia |
| ZIP/Postal code |
26506-9177 |
| Country |
USA |
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| Platform ID |
GPL13112 |
| Series (1) |
| GSE34483 |
H2A.Z Facilitates Access of Active and Repressive Complexes to Chromatin in Embryonic Stem Cell Self-renewal and Differentiation |
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| Relations |
| SRA |
SRX111899 |
| BioSample |
SAMN00765735 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM849958_GA2804-CMT1-shLuc-a-MNase-0.1U_r520l1.bed.gz |
168.2 Mb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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