|
| Status |
Public on Jul 15, 2021 |
| Title |
CMS-seq-INPUT |
| Sample type |
SRA |
| |
|
| Source name |
mesenchymal stem cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: umbilical cord-derived mesenchymal stem cells (PCS-500-010, lot #63216952)
age: PD12
antibody: N/A (input sample)
|
| 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 INPUT control for both replicate1 and replicate 2
|
| 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
|
| |
|
| 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 |
SAMN18108469 |
| SRA |
SRX10206598 |
