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| Status |
Public on Jun 22, 2022 |
| Title |
PolyA_plus_Don003_d10_rep2 |
| 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 10
donor id: Don003
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| Growth protocol |
Fresh blood was sourced either as 100 ml 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 Phase I mediumfor a three-phase differentiation adapted from that used for the BEL-A cell line (Trakarnsanga, Nat Comms 2017). Briefly, for differentiation 0.5-2.5x105 cells were resuspended on day 0 in Phase I media at 105 cells ml-1. Cell counts were performed on days 3 and 5 with additional Phase I media added to return the concentration to 105 cells ml-1. On day 7, cells were counted and pelleted (400 rcf, 5 min, RT) and resuspended in Phase II media at 3x105 cells ml-1. Cells were counted on day 9 and diluted to 3x105 cells ml-1 Phase II media. On day 11, cells were counted and pelleted (400 rcf, 5 min, RT) and resuspended in Phase III media at 106 cells ml-1. Cells were counted on days 13 and 15 and diluted to 106 cells ml-1 in Phase III media. Reproducibility between differentiations was confirmed morphologically with cytospins and immunologically with six FACS cell surface markers (CD34, CD36/Fatty acid translocase, CD71/Transferrin receptor, CD235a/Glycophorin A, CD49d/α-Integrin, CD233/Band).
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
isolation protocol: 1-5x10^6 cells were fixed in 1 ml TRI-reagent (Sigma), snap frozen and stored at -80ºC for less than one year.
RNA was extracted by addition of 0.1 ml 1-bromo-3-chloropropane, pipette mixing and separation in a Phase Lock gel Heavy tube (5Prime) and then precipitation with 1 µl of GlycoBlue and an equal volume (~500 µl) isopropanol and centrifugation (10 min, 12,000 rcf, 4 ºC). The RNA pellet was washed with 75 % ethanol, resuspended in DEPC-treated water, and stored at -80 ºC for less than one year.
For RNA-seq total RNA was treated with Turbo DNAse (Invitrogen) at 25 ºC for 60 min, then RNA was separated using phenol-chloroform isoamylalcohol and a PhaseLock Light-gel tube (5Prime). Treated RNA was precipitated at -80 ºC overnight with sodium acetate, glycoblue, and 75 % ethanol, before centrifugation (12,000 rcf, 4 ºC), 75 % ethanol wash and resuspension in DEPC-treated water. Globin and rRNA sequences were depleted from up to 5 µg of treated RNA using Globin-Zero Gold (Illumina), before PolyA selection with NEBNext Poly(A) mRNA Magnetic Isolation module (New England Biolabs), and indexing with NEBNext Ultra Directional RNA Library Prep Kit for Illumina (New England Biolabs) following the manufacturers’ instructions.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
Mapping: STAR --runThreadN 4 --outFilterMultimapNmax 1
Duplicate removal: samtools rmdup
Genome_build: hg19
Supplementary_files_format_and_content: bigwig: forward reads (deeptools) - bamCoverage -p4 --binSize 1 --normalizeUsingRPKM --filterRNAstrand forward
Supplementary_files_format_and_content: bigwig: reverse reads (deeptools) - bamCoverage -p4 --binSize 1 --normalizeUsingRPKM --filterRNAstrand reverse
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| Submission date |
Jan 30, 2019 |
| Last update date |
Jun 22, 2022 |
| 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) |
| GSE125924 |
Characterization of human erythropoiesis using ex vivo differentiation of CD34 HSC (RNA-seq) |
| GSE125926 |
Characterization of human erythropoiesis using ex vivo differentiation of CD34 HSC |
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| Relations |
| BioSample |
SAMN10848697 |
| SRA |
SRX5313143 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3585078_Don003_d10_rep2_fwd.bw |
48.4 Mb |
(ftp)(http) |
BW |
| GSM3585078_Don003_d10_rep2_rev.bw |
47.3 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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