|
| Status |
Public on Oct 05, 2016 |
| Title |
AB20_Day 14_SEM-F_CD71+, CD235a+ |
| Sample type |
RNA |
| |
|
| Source name |
AB_Day 14_SEM-F_CD71+,CD235a+
|
| Organism |
Homo sapiens |
| Characteristics |
days cultured: 14
source: Adult PBMCs
population: CD71+,CD235a+
media: SEM-F
enrichment method: Flow Cytometry
cell type: AB-EB
|
| Treatment protocol |
AB- and CB-derived EBs were stained and sorted on a MoFlo II (Beckman Coulter) using anti–CD36-PE (BD Biosciences; for EBs cultured for between four and seven days), anti–CD71-FITC (Dako), anti–CD235a-APC (BD Biosciences; for EBs cultured between four and 12 days), and anti–CD235a-RPE (Dako; for EBs cultured for 14 days). At day 0, AB-, CB- and hiPSC-derived HSPCs were isolated using CD34-beads (Miltenyi). HiPSC-derived hematopoietic progenitors were also isolated using CD31 beads at day 0 to incorporate all possible sources of erythroid progenitors. As insufficient cell numbers were obtained for FACS sorting of EBs derived from hiPSCs, CD71 and CD235a-specific magnetic beads were used to isolate hiPSC-EB populations at days 7 and 14 respectively. AB-EBs were isolated using these beads for direct comparison with hiPSC-derived EBs. Maturation of EBs was monitored by staining cytospin preparations from 5x104 cells with Wright-Giemsa and assessed using light microscopy. To visualise hemoglobin expression, cytospins were stained with 1% O-dianisidine in methanol and counterstained with 10% Giemsa (BDH).
|
| Growth protocol |
Human primary differentiating EBs derived from adult peripheral blood lymphocyte cones (AB) (NHS Blood and Transplant) or cord blood (CB) were obtained with informed consent in accordance with the Declaration of Helsinki. EBs were cultured using a three-phase liquid culture system {Griffiths, 2012 #516} with modifications. HiPSCs were established from fibroblasts or hematopoietic cells transduced with OCT4, SOX2 and KLF4 {Yang, 2014 #522} and EBs were generated by modifying a published protocol {Dias, 2011} in medium optimized for hiPSC-derived EB culture (SEM-i).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA including miRNA was extracted using mirVana (Life Technologies) according to the manufacturer’s instructions. RNA quality and quantity was assessed using a Qubit fluorimeter (Life Technologies), a NanoDrop spectrophotometer (Thermo) and an Agilent Bioanalyzer 2100 (Agilent Technologies). RNA integrity numbers were in the range 8.7-10. Before target preparation for hybridization to Affymetrix Human Transcriptome 2.0 (HTA2.0) arrays (Affymetrix), RNA was treated with Turbo DNA-Free DNAse (Life Technologies).
|
| Label |
Biotin
|
| Label protocol |
The target was prepared from 100ng DNAsed total RNA for hybridization to Affymetrix GeneChip Human Transcriptome 2.0 ST microarrays (HTA2) using the Ambion WT protocol (Life Technologies) and Affymetrix labelling and hybridization kits (Affymetrix). Labelled DNA mean yield was 13.5 μg (minimum: 8.5 μg; maximum: 20.5 μg).
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| |
|
| Hybridization protocol |
HTA2 arrays were hybridized with 5 μg of labelled DNA according to manufacturer's instructions.
|
| Scan protocol |
The Affymetrix GeneChip Fluidics Station 450 was used to wash and stain the arrays with streptavidin–phycoerythrin, according to the standard protocol for eukaryotic targets (IHC kit, Affymetrix). Arrays were scanned with an Affymetrix GeneChip scanner 3000 at 570nm.
|
| Data processing |
Intensity values were determined using GeneChip Operating Software (Affymetrix)and normalized by the Robust Multiarray Average algorithm using Affymetrix Expression Console software. Statistical analysis of differential expression was conducted using the Linear Models for Microarray Data package from the Bioconductor suite in R (www.bioconductor.org). The B values, p-values, and fold changes were used to select differentially expressed (DE) genes reaching a minimum linear expression value of 100 in all replicates of at least one sample group (p ≤ 0.01, fold change (FC) ≥ 2, B > 2.945). Principal component analysis (PCA) was conducted and displayed using Python packages. Hierarchical clustering (HC) analysis was performed using Python SciPy. Heat maps were generated using Python matplotlib. More advanced consensus clustering was conducted using SMART and Bi-CoPaM algorithms in MATLAB, using Biclustering from the biclust package in R. Biopython to fetch 1kb of genomic DNA sequence upstream of the transcriptional start sites (TSS) of each gene in the cluster. MEME suite was used for transcription factor binding site (TFBS) analysis, seeking homology with motifs in the Jolma database. Differentially-expressed genes were examined for enriched functional ontologies using GeneCoDis.
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| |
|
| Submission date |
Feb 24, 2015 |
| Last update date |
Oct 05, 2016 |
| Contact name |
Alison T Merryweather-Clarke |
| E-mail(s) |
alison.merryweather-clarke@admin.ox.ac.uk
|
| Organization name |
NHSBT Oxford
|
| Lab |
Blood Research Laboratory
|
| Street address |
John Radcliffe Hospital, Headley Way, Headington
|
| City |
Oxford |
| ZIP/Postal code |
OX3 9BQ |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL17586 |
| Series (1) |
| GSE66260 |
Distinct gene expression programs during erythropoiesis from adult and cord blood progenitor cells compared to hiPSCs |
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