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Sample GSM1866966 Query DataSets for GSM1866966
Status Public on Nov 26, 2015
Title WGBS seq WT LSK BR1
Sample type SRA
 
Source name wild type LSK cells biological replicate (BR) 1
Organism Mus musculus
Characteristics cell type: LSK cells
genotype: Wild Type
Treatment protocol bone marrow cells were flushed out of femurs and tibiae in Hanks' balanced salt solution (HBSS, Gibco Invitrogen) containing 2% FBS and 10 mM HEPES (Gibco Invitrogen). Prior to sorting of LSK cells in the bone marrow, we depleted lineage positive cells as follows. Red cell-depleted total bone marrow cells were incubated with Biotin-conjugated Mouse Hematopoietic Lineage Panel (eBioscience) at 4oC for 20 min. After washing with 1X PBS containing 1% FBS, cells were incubated with Dynabeads® Biotin Binder (Life Technologies) at 4oC for 30 min and Lin- cells were purified by magnetic separation, followed by staining with antibodies against c-Kit, Sca-1 and PerCP-Cy5.5 and sorting using FACSAria II.
Growth protocol Mice were housed in a pathogen-free animal facility at the La Jolla Institute for Allergy and Immunology and were used according to protocols approved by the Institutional Animal Care and use Committee
Extracted molecule genomic DNA
Extraction protocol After isolation of the cells, DNA was isolated using the PureLink DNA isolation kit (Life Technologies), according to the instructions of the manufacturer.
For WGBS library construction, genomic DNA was fragmented using a Covaris sonication system (Covaris S2). DNA libraries were constructed using the Illumina TruSeq Nano DNA sample preparation kit. Adapter ligated libraries were bisulfite-treated using the EpiTect Bisulfite Kit (Qiagen, Valencia, CA). Enrichment PCR was done using TrueSeq primer mix and Pfu TurboCx hotstart DNA polymerase (Stratagene).
 
Library strategy Bisulfite-Seq
Library source genomic
Library selection RANDOM
Instrument model Illumina Genome Analyzer IIx
 
Description bisulfite conversion
Data processing Fastq files from sequiencing were mapped against the mm9 genome using BSmap v2.74 and the parameters -v 4 -w 2 -r 0 -q 20 -R -p 12
To remove multiple mappers as well as methylation bias, BSeQC v1.0.3 with default parameters and -l 101_101 (Read length 100)
For each CpG covered by a given sample, the levels of methylation were calculated using methratio.py, script from BSmap tool with the parameters -z -t 0 -x CG -i "correct"
The methratio calling from all samples was merged into one big table (The MasterTable) containig all CpGs covered by at least one read in any of the samples.
Genome_build: mm9
Supplementary_files_format_and_content: Tabular format text, Mastertable, containing all the methylation info for the 6 samples (3 WT & 3 Tet2/3 DKO). The mastertable contains the CpG coverage and the total reads indication methylation in a given CpG for all samples. If a sample does not cover this CpG this is indicated with a NA in the methylation column and with zero coverage
 
Submission date Sep 02, 2015
Last update date May 15, 2019
Contact name Anjana Rao
E-mail(s) arao@lji.org
Phone 8587526500
Organization name La Jolla institute
Department Signaling and Gene Epression
Street address 9420 Athena Circle
City La Jolla
State/province CA
ZIP/Postal code 92037
Country USA
 
Platform ID GPL11002
Series (2)
GSE72629 Acute loss of TET function results in aggressive myeloid cancer in mice [WGBS]
GSE72630 Acute loss of TET function results in aggressive myeloid cancer in mice
Relations
BioSample SAMN04026697
SRA SRX1177958

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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