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Sample GSM2371309 Query DataSets for GSM2371309
Status Public on Jan 01, 2018
Title EZH1 KO AGM count
Sample type SRA
 
Source name Mouse VE-cadherin+cKit+CD45+CD41+ AGM cells
Organism Mus musculus
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 GPL17021
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 SAMN05967264
SRA SRX2319373

Supplementary file Size Download File type/resource
GSM2371309_EZH1_KO_AGM_count.txt.gz 100.5 Kb (ftp)(http) TXT
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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