|
| Status |
Public on Jul 15, 2021 |
| Title |
ucMSC-TL-Seq-Young |
| Sample type |
SRA |
| |
|
| Source name |
mesenchymal stem cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: umbilical cord-derived mesenchymal stem cells (PCS-500-010, lot #63216949)
cell passage: PD12
group: Young
|
| 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 |
polyA RNA |
| Extraction protocol |
DECAP-seq was performed as described in ref. 18 with some modifications. The starting material was poly-A+ RNA was isolated from 100mg of total RNA using the Dynabeads mRNA Purification Kit (Life Technologies, 61006). 5’ phosphate groups were removed using CIP (NEB, M0290) rather than AP, and Cap-Clip acid pyrophosphatase (CellScript, C-CC15011H) was used in place of RppH to de-cap the mRNAs.
Size selection of products was performed after 5’ adapter ligation, rather than after 1st strand synthesis to select against cDNAs generated solely from binding of the RT primer to the 5’ adapter. A negative control library was generated by omitting the de-capping step
TL-seq signal tracks were generated by bamCompare
|
| |
|
| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Data processing |
Raw reads were trimmed to remove sequencing adaptors and low quality reads using Trim Galore version 0.4.4 with default parameters.
TL-seq reads were aligned to the hg19 genome assembly using using STAR version 2.7 with default parameters.
Mapped reads on forward and reverse stands were separated by samtools view function. Reads from read2 were filtered due to the nature of the sequencing technology.
Cryptic TSSes (cTSSes) were identified in 10bp bins across the genome by comparing sample data to the negative control. Read count assessment was performed using the csaw package version 1.20 and the count data was normalized using the TMM method in edgeR version 3.28.
Signal tracks were generated with deeptools bamCompare version 3.2.0 with parameters: --operation subtract --binSize 10 --minMappingQuality 30
Genome_build: hg19
Supplementary_files_format_and_content: normalized signal tracks in bigwig format
|
| |
|
| Submission date |
Aug 18, 2020 |
| 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) |
| GSE156408 |
Age-associated cryptic transcription in mammalian stem cells is linked to permissive chromatin at cryptic promoters (TL-Seq) |
| GSE156409 |
Age-associated cryptic transcription in mammalian stem cells is linked to permissive chromatin at cryptic promoters |
|
| Relations |
| BioSample |
SAMN15848275 |
| SRA |
SRX8961873 |
