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| Status |
Public on Jul 08, 2020 |
| Title |
ATAC_Don003_Ery_d13_P1_rep1 |
| Sample type |
SRA |
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| Source name |
CD34 Culture
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| Organism |
Homo sapiens |
| Characteristics |
cell type: Erythroid
day of differentiation: Day 13
gender: Male
differentiation protocol: P1
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| Growth protocol |
Fresh whole blood collected from three healthy donors (two males, one female) using EDTA Vacuettes (Becton Dickson) or 5 ml leukocyte cones (NHS Blood & Transport). Whole cell counts were performed on a Pentra ES60 (Horiba) for donor blood to ensure clinically healthy red blood cell counts. Blood was diluted with PBS and overlaid onto Histopaque-1077 (Sigma) and centrifuged for 30 min at 630 rcf (no brake). Peripheral Blood Mononuclear Cells (PBMCs) were washed in PBS and MACS buffer (PBS, 2 µM EDTA, 0.5% BSA) and stained with Human CD34 Microbead kit (Miltenyi Biotec) following the manufacturer’s instruction for 30 minutes (4 ºC) before being passed successively through two LS Columns (Miltenyi Biotec) with three MACs buffer washes. Counting of cells was performed on a Luna FL (Logos) after staining with acridine orange (AO) and propidium iodide (PI). CD34+ cells were either stored in freezing buffer (90% FBS, 10% DMSO) or resuspended in growth media for differentiation. Protocol 1 (P1) was performed as per Mettananda et al. 2018 (PMID: 28871148): CD34+ cells were differentiated into erythroid cells over 21 days using a two-phase liquid culture system which used StemSpan SFEM II (Stemcell Technologies). Phase 1 medium was supplemented with stem cell factor, interleukin-3, human recombinant erythropoietin (EPO) (0.5 U/ml) and cholesterol-rich lipids. Phase 2 medium was similar to phase 1 medium except for addition of iron saturated holotransferrin and higher concentration of EPO (3 U/ml). Protocol 2 (P2) was performed as per Palii et al. 2011 (PMID: 21785407) and Palii et al 2019 (PMID: 30880026). The first step (Day0 to Day11) consists of cultivating CD34+ cells in serum-free IMDM medium supplemented with 1% penicillin/streptomycin, 4x10-3 ML glutamine, 40 ug/ml inositol, 10 ug/ml folic acid, 1.6x10-4 M monothioglycerol, 90 ng/ml ferrous nitrate, 900 ng/ml ferrous sulfate, 20% albumin-insulin-transferrin (BIT), also containing the following cytokines: 10-6 M hydrocortisone (HC), 100 ng/ml stem cell factor (SCF), 5 ng/ml interleukin 3 (IL-3) and 3 IU/ml erythropoietin (EPO) for 8 days followed by 3 days in supplemented IMDM medium containing only SCF and EPO. For the second step (Day12 to Day14), cells were co-cultured on a layer of stromal MS-5 cells in the supplemented IMDM medium containing only EPO. For the third step (Day15 to Day18), cells were co-cultured on a layer of MS-5 cells in the supplemented IMDM medium with no cytokines. For the fourth step (Day19 to Day24), cells were co-cultured on a layer of MS-5 cells in the supplemented IMDM medium in the presence of 10% fetal bovine serum.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Assay for transposition of active chromatin sequencing (ATAC-Seq) was performed as previously published (Buenrostro, 2013) on single cell suspensions. 60000-80000 cells were used per biological replicate. Cells were lysed and nuclei were isolated prior to transposition with Tn5 transposase (Nextera, Illumina) for 30 minutes at 37°C. DNA was purified using a MinElute kit (Qiagen).
Libraries were amplified and barcoded using the NEBNext 2xMastermix (NEB) and the custom primers as published in Buenrostro et al., 2013.ATAC-Seq libraries profiles were visualized using D1000 tape on the Tapestation (Agilent). The libraries were quantified using the universal library quantification kit (KAPA Biosystems).
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| Library strategy |
ATAC-seq |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
Genome-wide library of transposed fragments
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| Data processing |
Mapping : bowtie -m 2 --maxIns 350, mm9
For un-mapping reads : trimming adaptors trim_galore -- length 10, combining overlapping R1-R2 reads : flash -m 9 -x 0.125
Combining trimmed+flashed and originally mapped reads, mapping again with bowtie -m 2 --maxIns 350
Removing read pairs overlapping Blacklisted regions
Removing duplicates : samtools rmdup
Reconstructing sequenced fragments from R1+R2 read pairs ( bedtools bedpe | cut -f 1,2,6) - for visualisation
Pileup of fragments (bedtools genomecov -counts)
Generating bigwig tracks (deeptools bamCoverage --bam ${bamname}.bam -o ${bamname}.bw --binSize 10 --normalizeUsingRPKM --minMappingQuality 1)
Genome_build: hg19
Supplementary_files_format_and_content: bigWig : FPKM normalised coverage of filtered fragments (reconstructed, duplicate-filtered fragments)
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| Submission date |
Sep 05, 2019 |
| Last update date |
Jul 08, 2020 |
| Contact name |
Damien Downes |
| E-mail(s) |
damien.downes@ndcls.ox.ac.uk
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| Phone |
01865222374
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| Organization name |
The University of Oxford
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| Department |
MRC Weatherall Institute of Medicine
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| Street address |
John Radcliffe Hospital
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| City |
Headington |
| State/province |
Oxfordshire |
| ZIP/Postal code |
OX3 9DU |
| Country |
United Kingdom |
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| Platform ID |
GPL18573 |
| Series (2) |
| GSE136976 |
ATAC-seq from erythroid cells generated through ex vivo differentiation |
| GSE137983 |
Characterization of erythroid cells generated through ex vivo differentiation |
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| Relations |
| BioSample |
SAMN12703047 |
| SRA |
SRX6809258 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4064227_ATAC_Don003_Ery_d13_P1_rep1.bw |
167.5 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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