|
| Status |
Public on Apr 07, 2017 |
| Title |
CD33_CORRECTED_2 |
| Sample type |
RNA |
| |
|
| Source name |
CD33 sort, gene correction, replicate 2
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: iPSC-derived CD33+ promyelocyte
genotype/variation: HAX1corrected
|
| Treatment protocol |
iPSC-derived CD33-positive cells were purified by FACS sorting and collected in RNA Lysis Buffer (Zymo Research).
|
| Growth protocol |
iPSC colonies were detached as small fragments and shaken in suspension culture plates in Knockout Dulbecco's Modified Eagle Medium supplemented with 20 % Knockout serum replacement, 1 % nonessential amino acids, 1 % L-glutamine (all Thermo Scientific) at 100 rpm for 5 days. Embryoid bodies were then collected and transferred to adherent culture plates in StemDiff APEL medium (StemCell Technologies) supplemented with 50 ng/ml G-CSF and 25 ng/ml IL-3.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Quick-RNA™ MicroPrep (Zymo Research)
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 0.5 ug RNA using the One-Color Low RNA Input Linear Amplification PLUS kit (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
|
| |
|
| Hybridization protocol |
1.5 ug of Cy3-labelled cRNA (specific activity >10.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 250 ml containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 250 ml of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Whole Human Genome Oligo Microarrays (G4112A) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), then dried immediately by brief centrifugation.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505B) using one color scan setting for 1x44k array slides (Scan Area 61x21.6 mm, Scan resolution 10um, Dye channel is set to Green and Green PMT is set to 100%).
|
| Description |
gene expression of CD33+-sorted cells after promyelocytic differentiation of iPSCs
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 10.5 (Agilent) using default parameters to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
|
| |
|
| Submission date |
Apr 05, 2017 |
| Last update date |
Apr 07, 2017 |
| Contact name |
Jan-Henning Klusmann |
| E-mail(s) |
jan-henning.klusmann@kgu.de
|
| Organization name |
Goehte University Frankfurt
|
| Department |
Pediatric Hematology and Oncology
|
| Street address |
Theodor-Stern-Kai 7
|
| City |
Frankfurt |
| ZIP/Postal code |
60590 |
| Country |
Germany |
| |
|
| Platform ID |
GPL21185 |
| Series (1) |
| GSE97414 |
Gene profiling of HAX1-deficient iPSC-derived CD33-positive promyelocytes and their genetically corrected and wid-type counterparts |
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