|
Status |
Public on Jul 15, 2021 |
Title |
Young-CMS-seq-IP-rep2 |
Sample type |
SRA |
|
|
Source name |
mesenchymal stem cells
|
Organism |
Homo sapiens |
Characteristics |
cell type: umbilical cord-derived mesenchymal stem cells (PCS-500-010, lot #63216950) age: PD12 antibody: anti-CMS antibody described in PMID: 21552279
|
Treatment protocol |
Cells were cultured to PD12 and PD32.
|
Growth protocol |
Human cord blood mesenchymal stem cells (hMSCs) were purchased from ATCC (PCS-500-010, lot #63216949). Cells were grown in low glucose DMEM (Life Technologies 11885-084) supplemented with 10% FBS (Life Technologies 16000-044, lot #1314735) and 1× penicillin/streptomycin (Life Technologies 15140-122) at 37ºC with 5% CO2 and 3% O2. Medium was replaced every 4 days in the absence of passaging. Cells were grown to ~70% confluence and split 1:4 as needed. Cell density was below the concentration needed for reliable detection with a hemocytometer (105 cells/mL), so the growth curve was estimated as 2 population doublings per passage.
|
Extracted molecule |
genomic DNA |
Extraction protocol |
Genomic DNA was extracted from approximately 1 million hMSCs using the DNeasy Blood and Tissue kit (Qiagen #69504) and was sonicated to ~300bp with a Covaris focused ultrasonicator. Sonicated genomic DNA was ligated to methylated NEBNext Illumina adaptors using the NEBNext Ultra II DNA Library Prep (NEB, #E7645). Following bisulfite conversion with the EZ DNA methylation-lightning kit (Zymo Research, #D5030), samples were incubated with an anti-CMS antibody (from PMID: PMC3124347) bound to protein A/G Dynabeads (Thermo Fisher #10003D and #10001D). ChIP'ed DNA underwent phenol/chloroform/isoamyl acid cleanup and precipitation. Libraries were amplified for 10 PCR cycles with KAPA HiFi Uracil+ polymerase (KAPA Biosystems, #KK2801).
|
|
|
Library strategy |
OTHER |
Library source |
genomic |
Library selection |
other |
Instrument model |
Illumina HiSeq 2500 |
|
|
Description |
CMS-seq IP data replicate2 of young MSC
|
Data processing |
Library strategy: CMS-seq Raw reads were trimmed to remove sequencing adaptors and low quality reads using Trim Galore version 0.4.4 with default parameters. CMS-seq (5hmC) data were mapped using Bismark version 0.22.3. Peaks was called by MACS2 version 2.1.0 callpeak function using input as control, q-value < 0.01. Signaltracks were generated using macs2 bdgcmp function with -m ppois parameter. Genome_build: hg19 Supplementary_files_format_and_content: normalized signal tracks in bigwig format
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|
|
Submission date |
Mar 02, 2021 |
Last update date |
Jul 15, 2021 |
Contact name |
Weiwei Dang |
E-mail(s) |
weiwei.dang@bcm.edu
|
Organization name |
Baylor College of Medicine
|
Street address |
1 Baylor Plaza
|
City |
Houston |
State/province |
Texas |
ZIP/Postal code |
77030 |
Country |
USA |
|
|
Platform ID |
GPL16791 |
Series (2) |
GSE156409 |
Age-associated cryptic transcription in mammalian stem cells is linked to permissive chromatin at cryptic promoters |
GSE168064 |
Age-associated cryptic transcription in mammalian stem cells is linked to permissive chromatin at cryptic promoters [CMS-Seq] |
|
Relations |
BioSample |
SAMN18108472 |
SRA |
SRX10206595 |