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Sample GSM973531 Query DataSets for GSM973531
Status Public on Mar 18, 2013
Title iPS cell line 5hmC
Sample type SRA
Source name iPS cell line
Organism Mus musculus
Characteristics cell type: tet1 sox2 klf4 and c-Myc inducted iPS cells (derived driven from TSKM 2nd reprogramming system with full pluripotency)
stage or passages: p10-15
strain: OG2-GFP/Rosa26-M2rtTA
chip antibody: 5hmC pAb
chip antibody manufacturer: Active Motif
chip antibody catalog #: 39791
Treatment protocol The cells were cultured until the appropriate stage. The culture medium were threw out and washed with PBS. The cells were centrifuge dissociated by 0.05% trypsin EDTA.The ESCs were added 2-folds-volume ESM and dissociated to single cells. The cell suspension were plated in new dished for 0.5hr(37℃) to clear away the feeder cells. The supernatant supension were further collected carefully and centrifuged for RNA extraction. The fibroblasts were centrifuged to collect the cells and treated with centrifuge centrifuged and treated with Trizol (Invirtrogen).
Growth protocol The iPS cells were cultured on mitomycin C treated MEFs in ES medium containing DMEM medium (Gibco Invitrogen, Carlsbad, CA) supplemented with 15% (v/v) fetal bovine serum (FBS), 1 mM L-glutamine, 0.1 mM mercaptoethanol, 1% nonessential amino acid stock, and 1000 U/ml LIF. The TSKM 2nd induction cells were deriven from TSKM iPS mice and cultured with Feeder Medium for 3 passages. On passages 4 the culture were changed to ES Medium with doxycycline for 3 days.
Extracted molecule genomic DNA
Extraction protocol In brief, the sonicated genomic DNA was treated using NEBNext master Mix to repair the ends of DNA fragments and ligate the paired-end sequencing specific adaptors (Illumina), the DNA product was then immunoprecipitated and purified. Here we used 500ng ligation product for each IP reaction. Fragments were amplified with 14–16 cycles using adaptor specific primers (Illumina). The final product (300-500bp) was gel-purified (Qiagen) before cluster generation and sequencing.
Library strategy MeDIP-Seq
Library source genomic
Library selection 5-methylcytidine antibody
Instrument model Illumina HiSeq 2000
Data processing The reads generated by Illumina standard pipeline (CASAVA version 1.8) were mapped to mouse reference genome (mm9/NCBI37) using Bowtie (v0.12.7) software allowing the max mismatch 3nt. Only unique mapped reads were extracted for the following analysis.
The aligned reads were further feed to MACS (v1.4.1) software to identify the DNA modification enriched regions (peaks). The MACS parameters were referred to the literature (default parameters with addition of "--nolambda --nomodel -g mm")
To adjust the library size variance and facilitate the data exhibition, the total 30 million mapped reads were randomly selected from each sample.
Genome_build: mouse reference genome (mm9/NCBI37)
Supplementary_files_format_and_content: Bed files were generated using MACS (v1.4.1), it contained the genomic coordinates of identified peaks
Submission date Jul 25, 2012
Last update date May 15, 2019
Contact name Tao Cai
Organization name National Institute Of Biological Sciences, Beijing (NIBS)
Lab Sequencing Facility
Street address No. 7 Science Park Road, Zhongguancun Life Science Park
City Beijing
ZIP/Postal code 102206
Country China
Platform ID GPL13112
Series (2)
GSE39638 Genome-wide maps of 5hmC/5mC state in pluripotent and (T-)iPS induction cells.
GSE39639 Gene expression and 5hmC/5mC state in pluripotent and TSKM-iPS induction cells
SRA SRX170894
BioSample SAMN01093929

Supplementary file Size Download File type/resource
GSM973531_TSKMJ-3_5H_peaks.bed.gz 2.4 Mb (ftp)(http) BED
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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