|
| Status |
Public on May 13, 2015 |
| Title |
m_KO_hMSC |
| Sample type |
SRA |
| |
|
| Source name |
Mesenchymal stem cell
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: Mesenchymal stem cell
genotype/variation: KO
|
| Treatment protocol |
We sought to generate an isogenic human embryonic stem cell (hESC) line by knocking out the conserved 15th and 16th exons of WRN gene encoding for the DNA helicase domain of the WRN protein by helper-dependent adenoviral vector (HDAdV).
|
| Growth protocol |
H9 hESCs (WiCell Research) and their WRN mutant derivatives were maintained on Mitomycin C treated mouse embryonic fibroblasts (MEFs) in hESC medium or on Matrigel (BD Biosciences) coated plates in mTeSR medium (STEMCELL Technology). Human bone marrow-derived MSCs were maintained in Minimum Essential Medium (MEM) Alpha Medium supplemented with 10% FBS, and 0.1 mM NEAA with or without 1ng/ml bFGF.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Total DNA was extracted from the cells using the DNeasy Blood & Tissue Kit (Qiagen) following the manufacturer’s protocol
For RRBS, 100-200ng of genomic DNA was digested with Msp I and purified by QIAquick PCR Purification kit (Qiagen)QIAquick kit. 50-600bp fragments were selected by 2% agarose gel. After addition of spike-in controls, samples were end-repaired, A-tailed and ligated to methylated adapters employing TruSeq DNA Sample Preparation Kit (Illumina). The ligated DNA was then treated with bisulfate using MethylCode Kit (Invitrogen) converting unmethylated cytosine (C) into uracil (U). Finally, sequencing libraries were prepared by PCR amplification with PfuTurbo CĂ— Hotstart DNA polymerase (Agilent Technologies).
|
| |
|
| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
Reduced Representation |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
Total DNA extracted from cells
RRBS
processed data file: hMSC-KO-RRBS-TARRBS.bed.gz
|
| Data processing |
Illumina CASAVA version 1.8 were used to the basecalling.
Reads were trimmed to remove the adapter sequences and low quality bases.
Clean reads were aligned to gynab refference genome (hg19) using BWA software
RPKM of each gene was determined by the following formula: RPKM=TR/(MR(million)*EL(KB)), TR is the total reads that mapped to the reference, MR is total number of the reads that mapped to one of the transcripts of the gene, EL is the length of the longest transcript of a gene.
Unique mapping reads were used to call peaks by MACS(v1.4) software
Genome_build: hg19
Supplementary_files_format_and_content: [RNA-Seq] txt files which include the RPKM information of each RNA-Seq sample
Supplementary_files_format_and_content: [ChIP-Seq] bed files which include the peaks called by MACS(v1.4)
Supplementary_files_format_and_content: [ChIP-Seq] bedgraph files which depict the reads distribution among the genome
Supplementary_files_format_and_content: [RRBS-Seq] bed files which include the C and C+T of RRBS
|
| |
|
| Submission date |
Apr 07, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Jingyi Li |
| E-mail(s) |
lijy201@gmail.com
|
| Organization name |
Peking University
|
| Street address |
Yiheyuan Road, No.5, Haidian District
|
| City |
Beijing |
| ZIP/Postal code |
100871 |
| Country |
China |
| |
|
| Platform ID |
GPL11154 |
| Series (1) |
| GSE52285 |
A Werner syndrome stem cell model unveils heterochromatin alterations as a driver of human aging |
|
| Relations |
| BioSample |
SAMN02720870 |
| SRA |
SRX512181 |
