|
Status |
Public on Jul 24, 2014 |
Title |
MYB prom donor A |
Sample type |
SRA |
|
|
Source name |
human erythroid progenitor (HEP)
|
Organism |
Homo sapiens |
Characteristics |
cell type: human erythroid progenitor (HEP) treatment: MYB promoter
|
Growth protocol |
Erythroid cells were cultured using a 2-phase liquid system. Mononuclear cells were isolated from peripheral blood by centrifugation on a gradient of Ficoll-Hypaque and cultured for 7 days in phase I medium which consists of serum-free StemSpan (Stem Cell Technologies) supplemented with 1 µg/mL cyclosporin A, 25 ng/mL interleukin-3, 50 ng/mL human stem cell factor (Sigma), and 0.01% bovine serum albumin. Cells were incubated at 37°C, 5% CO2. After 7 days, nonadherent cells were collected and reseeded at a concentration of 2.5 x 105 cells/mL in phase II medium (StemSpan supplemented with 10-7 M dexamethasone σ, 50 ng/mL stem cell factor, and 2 U/mL human recombinant erythropoietin [EPO; Sigma]). The cultures were diluted once or twice to maintain the cell concentration lower than 1 x 106 cells/mL in phase II. Cell samples were collected from phase II cultures on days 0, 2, 3, 4, 5, 6, and 7 and evaluated for number and viability.
|
Extracted molecule |
genomic DNA |
Extraction protocol |
Cells were harvested at day 7-8 of culture and crosslinked using formaldehyde (1% final volume for ChIP-Seq, 2% final volume for 3C-Seq) and nuclei were isolated. For ChIP-Seq, nuclei were sonicated and protein-DNA complexes isolated using antibodies. For 3C-Seq, nuclei were digested and ligated under dilute conditions with primary and secondary restriction enzymes. ChIP-Seq libraries were prepared for sequencing using standard Illumina protocols. 3C-seq library preparation was described in detail in Stadhouders et al. Nature Protocols (2013).
|
|
|
Library strategy |
OTHER |
Library source |
genomic |
Library selection |
other |
Instrument model |
Illumina Genome Analyzer |
|
|
Description |
3C-Seq
|
Data processing |
Basecalls performed using CASAVA version 1.6 (Bustard) Alignment performed using Bowtie against hg18 reference genome Only unique mapping reads were used for the ChIP-seq downstream analysis Genome_build: hg18 Supplementary_files_format_and_content: BigWig
|
|
|
Submission date |
Nov 21, 2013 |
Last update date |
May 15, 2019 |
Contact name |
Supat Thongjuea |
E-mail(s) |
supat.thongjuea@ndcls.ox.ac.uk
|
Organization name |
The Weatherall Institute of Molecular Medicine
|
Department |
MRC Molecular Haematology Unit
|
Street address |
Headington
|
City |
Oxford |
ZIP/Postal code |
OX3 9DS |
Country |
United Kingdom |
|
|
Platform ID |
GPL9052 |
Series (1) |
GSE52637 |
HBS1L-MYB intergenic variants modulate fetal hemoglobin via long-range MYB enhancers |
|
Relations |
BioSample |
SAMN02419109 |
SRA |
SRX381547 |