A1
Homo sapiens
Protocols: Hematopoietic differentiation: We used a two-dimensional “monolayer” system to differentiate human pluripotent stem cells (iPSCs) into “primitive” hematopoietic progenitor cells (hHPCs) as described.(Mills et al. 2014) On day -1, iPSCs were cultured in 100 x 20 mm petri dishes in the presence of 0.5 mM EDTA to reach a 5-10% confluency. On day 0, StemMACS iPS-Brew XF medium was replaced by complete hematopoietic differentiation medium supplemented with 5 ng/ml BMP4 (Peprotech, Cat. No. 120-05), 50 ng/ml VEGF165 (Peprotech, Cat. No. 100-20) and 1 μM GSK-3β inhibitor (CHIR99021, Sigma Aldrich, Cat. No. SML1046-5MG). On day 2, this medium was replaced by hematopoietic differentiation medium [Serum free medium(Casadevall et al. 1982) mixed 1:1 with complete StemPro34 medium and supplemented with 1% nonessential amino acids, 1mM L-glutamine, 0.5mg/ml Penicillin-Streptomycin, 0.1mM β-Mercaptoethanol, 1% Insulin-Transferrin-Selenium (ThermoFisher, Cat. No. 41400045), 10% bovine serum albumin (BSA, Hyclone) fraction V, and 50 μg/ml ascorbic acid (Sigma Aldrich, Cat. No. A4403)]. This medium was further supplemented with 5ng/ml BMP4, 50ng/ml VEGF165 and 20ng/ml of bFGF. This medium was replaced on day 4 by hematopoietic differentiation medium supplemented with 50ng/ml VEGF165 and 5ng/ml bFGF, on day 6 by hematopoietic differentiation medium supplemented with 50ng/ml VEGF165, 50ng/ml bFGF, 50ng/ml SCF, and 5ng/ml Flt3LG, and on day 8 by hematopoietic differentiation medium supplemented with 50 ng/ml VEGF165, 50ng/ml bFGF, 50ng/ml SCF, 5ng/ml Flt3LG, 10ng/ml Interleukin-6 (IL-6, Peprotech, Cat. No. 200-06) and 50ng/ml thrombopoietin (TPO, kindly provided by Kirin, Japon). hHPCs sorted at day 10-16. Total RNA was isolated from primary CD34+ cells with TriZol (Invitrogen). The RNA integrity (RNA Integrity Score ≥ 7.0) was checked on the Agilent 2100 Bioanalyzer (Agilent) and quantity was determined using Qubit (Invitrogen). SureSelect Automated Strand Specific RNA Library Preparation Kit was used according to manufacturer's instructions with the Bravo Platform. Briefly, 50 ng of total RNA sample was used for poly-A mRNA selection using oligo(dT) beads and subjected to thermal mRNA fragmentation. The fragmented mRNA samples were subjected to cDNA synthesis and were further converted into double stranded DNA using the reagents supplied in the kit, and the resulting dsDNA was used for library preparation. The final libraries were processed using Illumina TruSeq Stranded mRNA Library Prep Kit.
ENA-FIRST-PUBLIC
2019-04-30T04:02:50Z
ENA-LAST-UPDATE
2019-04-09T16:04:33Z
External Id
SAMEA5560257
INSDC center name
IHPST (UMR8590), Universite Paris I-Pantheon Sorbonne UMR1170, Gustave Roussy Cancer Campus
INSDC first public
2019-04-30T04:02:50Z
INSDC last update
2019-04-09T16:04:33Z
INSDC status
public
Submitter Id
E-MTAB-7850:A1
broker name
ArrayExpress
cell type
CD34-positive, CD43-positive hematopoietic progenitors, CMML
common name
human
disease
chronic myelomonocytic leukemia
sample name
E-MTAB-7850:A1
scientific_name
Homo sapiens