|
| Status |
Public on Jan 01, 2018 |
| Title |
MSC-iPS 5F +shEZH1 D6 day 4 rep 1 |
| Sample type |
SRA |
| |
|
| Source name |
Human MSC iPS-derived 5F hematopoietic cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: Hematopoietic cells
|
| Treatment protocol |
5F cells were transfected with 2x shRNAs targeting the luciferase (shRNAs F8 and C11) and EZH1(shRNAs D4 and D6) genes. mRNA was collected 4 and 28 days after transfection
|
| Growth protocol |
Human CD34- and MSC-iPS cells were differentiated to CD34+ hematopoietic cells and transduced with 5 transcription factors (5F). Human 5F cells were cultured in SFEM with 50 ng/ml SCF, 50 ng/ml FLT3, 50 ng/ml TPO, 50 ng/ml IL6, and 10 ng/ml IL3 (all R&D Systems). Dox was added at 2 μg/ml (Sigma) and puromycin was added at 0.35 ug/mL (ThermoFisher). Media were changed every 3-4 days. RNA was harvested from bulk day 4 samples, or from sorted CD34+CD38- day 28 samples. Mouse AGM and YS were microdissected from E10.5 embryos and dissociated to single cells. Cells were then sorted for immunophenotypic markers cKit+VE-Cadherin+CD45+CD41+.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
For 5F cells, we used the NEB ultradirectional kit with 50 ng of input RNA. For mouse YS and AGM cells, we used the Clontech smarterseq kit with 10 ng input RNA.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Data processing |
From the raw reads, we removed adaptor sequences using a Cutadapt tool, allowing some mismatches (The specific parameters we used were -n 2 -e 0.1 –o 10)
Then, the cleaned reads were aligned to the human genome/transcriptome (hg19 and corresponding UCSC gene model) using a TopHat2 software with the following parameters (--library-type=fr-unstranded --min-intron-length=10 --no-coverage-search --microexon-search --min-isoform-fraction=0).
To estimate expression levels for each gene, we counted aligned reads per gene using a htseq-count tool with the following parameters (--stranded=no --idattr=gene_id -t exon -i gene_name).
Genome_build: hg19
Supplementary_files_format_and_content: .txt files containing read counts from htseq-count
|
| |
|
| Submission date |
Nov 02, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Melissa Kinney |
| E-mail(s) |
melissa.kinney@childrens.harvard.edu
|
| Organization name |
Boston Children's Hospital
|
| Street address |
1 Blackfan Circle
|
| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02215 |
| Country |
USA |
| |
|
| Platform ID |
GPL16791 |
| Series (2) |
| GSE89415 |
RNA-seq analysis of EZH1 knockdown in 5F cells, or EZH1 heterozygous and homozygous knockout YS and AGM cells |
| GSE89418 |
EZH1 as a key epigenetic barrier to definitive haematopoiesis during embryonic development |
|
| Relations |
| BioSample |
SAMN05967295 |
| SRA |
SRX2319343 |
