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| Status |
Public on May 26, 2020 |
| Title |
Runx1cPos_3Dox_Rep3 RNA-Seq |
| Sample type |
SRA |
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| Source name |
hESC H9 cell line
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| Organism |
Homo sapiens |
| Characteristics |
cell line: H9
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| Treatment protocol |
Hematopoietic differentiation of human ESCs was performed following the spin Embryo Body (EB) method in STAPEL medium with supplements used as reported by Ng et al (2016) Blood 120, 4038–4048. with some modifications, detailed below. Albumin was substituted for a 1:1 mixture of albumin from rice endosperm (ScienCell Research Labs cat# OsSA) and Bovostar acid stripped Bovine Serum Albumin (BSA, Bovogen cat# BSAS 0.1). For the first 4 days, 7.5 ng/ml ACTIVIN A (ACT, R&D Systems cat# 338-AC) and 0.5 μM CHIR99021 (Tocris Biosciences cat# 4423) were used and 3.5 µM SB431542 (Sapphire Bioscience cat# 13031) and 3 µM CHIR99021 were added 4-6 hours before the day 2 time point and until day 4. After day 4, 5 ng/ml BMP4 and 10 ng/ml Insulin-like growth factor 2 (IGF2, PeproTech cat# 100-12) were used instead of the original concentrations. Adherent cultures after d8 were supplemented with 20 ng/ml BMP4, 100 ng/ml stem cell factor (SCF, PeproTech cat# 300-07), 100 ng/ml FMS-like tyrosine kinase 3 receptor (FLT3) ligand (PeproTech cat# 300-19), 50 ng/ml Thrombopoietin (TPO, PeproTech cat# 300-18), 50 ng/ml vascular endothelial growth factor (VEGF, PeproTech cat# 100-20), 25 ng/ml Interleukin (IL) 3 (PeproTech cat# 200-03), 25 ng/ml IL6 (PeproTech cat# 200-06), 20 ng/ml IGF2, 10 ng/ml basic Fibroblast Growth Factor (FGF2, PeproTech cat# 100-18B) and 1x Penicillin/Streptomycin (Pen/Strep, 5000 U/ml and 5000 U/ml respectively, Thermo Fisher Scientific cat# 15140122). Plates were toped up with media every 3 days and half-media changes were performed when the media capacity of the plate was reached. Upon Dox treatment, adherent cultures were supplemented with a ‘5-factor’ cytokine mix including 100 ng/ml SCF, 100 ng/ml FLT3 ligand, 50 ng/ml TPO, 25 ng/ml IL3 and 25 ng/ml IL-6. For analysis, EBs were harvested at different time points and dissociated into single-cell suspensions using TryPLE select (Invitrogen) for non-adherent EBs and Collagenase Type 1 or Type 4 (Worthington, CLS-1 or CLS-4) for adherent EB cultures and passed through 23- and 25-gauge needles and a 40µm filter.
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| Growth protocol |
Culture and enzymatic passaging of hESC lines was conducted as previously reported by Ng et al. (2008) Nat. Protoc. 3, 768–776.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using the Bioline Isolate II RNA Mini Kit according to the manufacturers’ instructions. cDNA was reverse-transcribed using random hexamer priming and Tetro cDNA synthesis kit (Bioline) or using Oligo (dT)18 priming and SuperScript™ II Reverse Transcriptase (Thermo Fisher Scientific) according to the manufacturers’ instructions.
RNA-seq libraries were prepared using a TruSeq Stranded mRNA Library Prep (Illumina cat# 20020594) following the Low Sample (LS) workflow according to manufacturer’s instructions.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
Runx1cPos_3Dox_Rep3
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| Data processing |
Reads were processed with Trimmomatic v0.32 to remove low quality bases and sequencing adaptors
Processed reads were aligned to the human genome (hg38) using Hisat2 v2.1.0 with default parameters
Gene expression was quantified as Fragments Per Kilobase per Million mapped reads (FPKM) using Stringtie v1.3.3 with default parameters
Genome_build: hg38
Supplementary_files_format_and_content: Tab-delimited file containing gene symbols and FPKM values
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| Submission date |
Sep 18, 2019 |
| Last update date |
May 26, 2020 |
| Contact name |
Peter Keane |
| E-mail(s) |
[email protected]
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| Organization name |
University of Birmingham
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| Department |
Institute for Cancer and Genomic Sciences
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| Street address |
Vincent Drive
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| City |
Birmingham |
| ZIP/Postal code |
B15 2TT |
| Country |
United Kingdom |
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| Platform ID |
GPL18573 |
| Series (2) |
| GSE137671 |
Expression of RUNX1-ETO Rapidly Alters the Chromatin Landscape and Growth of Early Human Myeloid Precursor Cells (RNA-Seq) |
| GSE137673 |
Expression of RUNX1-ETO Rapidly Alters the Chromatin Landscape and Growth of Early Human Myeloid Precursor Cells |
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| Relations |
| BioSample |
SAMN12783616 |
| SRA |
SRX6867689 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4083992_Runx1cPos_3Dox_Rep3.tsv.gz |
209.7 Kb |
(ftp)(http) |
TSV |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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