|
| Status |
Public on Dec 31, 2020 |
| Title |
CD34+DNAM-1brCXCR4+ cells - progeny, biol rep 2 |
| Sample type |
RNA |
| |
|
| Source name |
CD34+DNAM-1brCXCR4+ progeny
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: NKG2C+ NK cells
cell subtype: CD34+DNAM-1brCXCR4+ progeny
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using TRIzol reagent and purified with RNeasy Micro kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer’s protocol
|
| Label |
biotin
|
| Label protocol |
The total RNA (10 ng) was reverse transcribed using GeneChip® WT Pico Reagent Kit (Affymetrix; Thermo Fisher Scientific, Inc.) following manufacturer’s protocol. The obtained antisense cRNA was purified using Nucleic Acid Binding Beads (GeneChip® WT Pico Reagent Kit, Affymetrix) and used as a template for reverse transcription to produce single-stranded DNA in the sense orientation. During this step, dUTP was incorporated. The DNA was then fragmented using uracil DNA glycosylase (UDG) and apurinic/apyrimidinic endonuclease 1 (APE 1) and labeled with DNA labeling reagent covalently linked to biotin using terminal deoxynucleotidyl transferase (TdT, GeneChip® WT Pico Reagent Kit, Affymetrix)
|
| |
|
| Hybridization protocol |
Hybridization of each fragmented and labeled target was performed using the GeneChip® Hybridization, Wash and Stain Kit (Affymetrix; Thermo Fisher Scientific, Inc). Targets were diluted in hybridization buffer at a concentration of 25 ng/ul and denatured at 99 °C for 5 minutes, incubated at 45 °C for 5 minutes and centrifuged at maximum speed for 1 minute prior to introduction into the GeneChip® cartridge. A single GeneChip® Human Clariom D was then hybridized with each biotin-labeled sense target. Hybridizations were performed for 16 h at 45 °C in a rotisserie oven (60 RPM). GeneChip® cartridges were washed and stained with GeneChip® Hybridization, Wash and Stain Kit in the Affymetrix Fluidics Station 450 following the FS450_0001 standard protocol.
|
| Scan protocol |
GeneChip arrays were scanned using an Affymetrix GeneChip® Scanner 3000 7G using default parameters. Affymetrix GeneChip® Command Console software (AGCC) was used to acquire GeneChip® images and generate .DAT and .CEL files
|
| Data processing |
The data were analyzed with Transcriptome Analysis Console v. 4.0 software (Thermo Fisher Scientific) using default Affymetrix configuration settings and gene-level SST-RMA summarization alghorithm.
|
| |
|
| Submission date |
Aug 28, 2019 |
| Last update date |
Jan 01, 2021 |
| Contact name |
Andrea Pelosi |
| E-mail(s) |
andrea.pelosi@opbg.net, pelosi.andrea81@gmail.com
|
| Phone |
+390668594418
|
| Organization name |
Ospedale Pediatrico Bambino Gesù
|
| Department |
Laboratories
|
| Lab |
Immunology
|
| Street address |
Viale di San Paolo, 15
|
| City |
Rome |
| State/province |
RM |
| ZIP/Postal code |
00146 |
| Country |
Italy |
| |
|
| Platform ID |
GPL23126 |
| Series (1) |
| GSE136570 |
Gene expression profiling of NKG2C+ NK cells generated from inflammatory CD34+DNAM-1brCXCR4+ and Lin-CD34- CD16+CD56- blood precursors |
|
