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Sample GSM849958 Query DataSets for GSM849958
Status Public on Dec 20, 2012
Title mES_Mnase_shluc_ctrl_for_H2AZKD
Sample type SRA
 
Source name mouse ES cells, shluc control, MNase
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
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.
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.
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.
 
Library strategy MNase-Seq
Library source genomic
Library selection MNase
Instrument model Illumina HiSeq 2000
 
Description Direct sequencing following MNase digestion on x-linked chromatin.
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).
 
Submission date Dec 15, 2011
Last update date May 15, 2019
Contact name Gangqing Hu
E-mail(s) michael.hu@hsc.wvu.edu
Organization name West Virginia University
Department MicroBiology, Immunology, and Cell Biology
Lab 2072A, HSC North, Floor 2
Street address 64 Medical Center Drive
City Morgantown
State/province West Virginia
ZIP/Postal code 26506-9177
Country USA
 
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
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 SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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