|
| Status |
Public on Dec 31, 2013 |
| Title |
immortalized erythroblasts (donor b) at day 141 of expansion culture (after viral transduction) |
| Sample type |
RNA |
| |
|
| Source name |
immortalized erythroblasts at day 141 of expansion culture (after viral transduction)
|
| Organism |
Homo sapiens |
| Characteristics |
tissue of origin: cord blood
expansion days: 19+141 days
genetic factors: Sox2, Klf4, c-Myc and p53shRNA
|
| Treatment protocol |
no treatment
|
| Growth protocol |
pCBE19: After thawing, frozen CB MNCs (5x106/ml) were cultivated in serum-free medium (SFM; 50% IMDM with 50% Ham’s F12 (Invitrogen), synthetic lipids (Invitrogen), insulin-transferrin-selenium supplement (Invitrogen) and 5 mg/ml BSA (Sigma), 50 ug/ml of ascorbic acid (Sigma) and 2 mM glutamax ) supplemented with Epo (2 U/mL; R&D systems), Dexamethasone (1 µM; Sigma), insulin-like growth factor 1 (IGF-1; 40 ng/mL), stem cell factor (SCF; 100 ng/mL), and holo-transferrin (100 µg/ml; Sigma), termed “erythroblast expansion medium” in gelatin-coated wells. Primary cultures of erythroblasts established after several days were kept less than 1x106 cells/ml by daily cell counting and partial medium changes. iE2: Grow in the erythroblast expansion medium. After the culture cells become proliferating constantly, only partial medium change and cell passaging every 2 or 3 days are performed to keep cell density at or below 5 × 105 cells/ml. CD34+: primary cells isolated by magenetic beads binding to CD34 cell surface marker. TF-1: RPMI-1640+10% FBS+ 3U/ml hEPO. iPSC and ESC: standard ESC medium and on MEF feeder.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNeasy according to manufacture's protocol
|
| Label |
biotin
|
| Label protocol |
One round amplification protocol for total RNA following Affymetrix’ specifications using T7 promoter engineered random primers for cDNA synthesis, and Ambion® WT Expression Kit (www.affymetrix.com).
|
| |
|
| Hybridization protocol |
16hrs at 45° C with rotation (60rpm) as described by Affymetrix in their GeneChip® Expression Wash, Stain and Scan User Manual (www.affymetrix.com).
|
| Scan protocol |
Using Affymetrix’ GeneChip Scanner 3000 7G and default parameters described by the manufacturer in their GeneChip Expression Analysis Technical Manual.
|
| Description |
immortalized erythroblasts at day 141 of expansion culture (after viral transduction) in serum-free medium supplemented with SCF, IL-3, IGF-1, EPO and Dexamethasone
iE2(d141)
|
| Data processing |
Image analysis used the Affymetrix Command Console version 3 (AGCC v3.0) software, processed with the Expression Console PLIER algorithm and the manufacturer’s specifications.
probe group file: HuGene-1_0-st-v1.r4.pgf
meta-probeset file: HuGene-1_0-st-v1.na33.1.hg19.probeset.csv, HuGene-1_0-st-v1.na33.1.hg19.transcript.csv
RMA Log2 values from the Partek Genomics Suite, using all probesets, were summarized for gene-level analysis.
|
| |
|
| Submission date |
Feb 07, 2013 |
| Last update date |
Dec 31, 2013 |
| Contact name |
Xiaosong Huang |
| E-mail(s) |
xhuang18@jhmi.edu
|
| Organization name |
Johns Hopkins University
|
| Department |
Medicine/Hematology
|
| Lab |
Linzhao Cheng
|
| Street address |
733 North Broadway, Rm780
|
| City |
Baltimore |
| State/province |
Maryland |
| ZIP/Postal code |
21205 |
| Country |
USA |
| |
|
| Platform ID |
GPL10739 |
| Series (1) |
| GSE44136 |
Expression data from primary CB erythroblasts, immortalized/induced erythroblasts, Fetal liver CD34+ blood stem cells, adult CD34+ blood stem cells, erythroleukemia cell line (TF-1) and human ESC and iPSCs |
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