|
| Status |
Public on Dec 07, 2023 |
| Title |
CD34+_APOBECYTH_STM2457 rep2 |
| Sample type |
SRA |
| |
|
| Source name |
cord blood
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: cord blood
cell type: primary cells
cell lineage: hematopoietic stem cell
construct: APOBEC-YTH OE
treatment: STM2457
|
| Treatment protocol |
Cells were infected with virus expressing APOBEC-YTH retrovirus or empty vector. 24hr post infection, CD34+ cells were treated with STM2457 at a concentration of 20µM for 2 days. GFP+ cells were sorted .
Sorted cells were harvested and frozen in TRIzol reagent. RNA was extracted using Phenol-Chloroform
|
| Growth protocol |
Human CD34+ HSPCs were purified from at least 6 mixed CB units (each unit from one healthy donor). Freshly purified CD34+ cells were cultured overnight in X-Vivo 10 media containing 1% BSA, 100ng/ml hSCF, 100ng/ml hFlt3L, 50ng/ml hTPO, and 10ng/ml hIL7, PeproTec.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
After PicoGreen quantification and quality control by Agilent BioAnalyzer, library preparation is performed using the SMART-Seq v4 Ultra Low Input RNA Kit
After PicoGreen quantification and quality control by Agilent BioAnalyzer, library preparation is performed using the SMART-Seq v4 Ultra Low Input RNA Kit
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
| |
|
| Data processing |
Adapter trimmer of the fastq files was performed using Cutadapt (v1.13) with option -m 50.
Aligment to human (hg38) genomes using HISAT3N78 (v2.2.1-3n) with –base-change C,T and –no-repeat-index as parameters.
Bullseye pipeline (https://github.com/mflamand/Bullseye) was employed to identify C-to-T mutations in each RNA-seq library. A minimal coverage of 10 reads and duplicate removal were required to construct the variant quantification matrices using the parseBAM.pl script, provided by the Bullseye pipeline. The Bullseye/Find_edit_site.pl was applied with parameters --ControlMinCoverage 10, --EditMinCoverage 10, --minEdit 2.5, --maxEdit 95, --editFoldThreshold 1, --bedt, --intron, and --extUTR. Additionally, --minEditSites 1 was used to account for edited sites that may appear as single positions instead of clusters.
Assembly: hg38
Supplementary files format and content: tab-delimited text files include read variant values per position.
|
| |
|
| Submission date |
May 07, 2023 |
| Last update date |
Dec 07, 2023 |
| Contact name |
Michael G. Kharas |
| E-mail(s) |
[email protected]
|
| Organization name |
Memorial Sloan Kettering Cancer Center
|
| Department |
Molecular Pharmacology Program
|
| Street address |
417 East 68th Street
|
| City |
New York |
| State/province |
NY |
| ZIP/Postal code |
10065 |
| Country |
USA |
| |
|
| Platform ID |
GPL20301 |
| Series (1) |
| GSE231861 |
DARTseq identifies m6A targets in human CD34+ HSPCs |
|
| Relations |
| BioSample |
SAMN34992367 |
| SRA |
SRX20248448 |
