|
| Status |
Public on Sep 09, 2017 |
| Title |
DWT-II+III [PJI-13] |
| Sample type |
RNA |
| |
|
| Source name |
DWT-II+III
|
| Organism |
Mus musculus |
| Characteristics |
strain background: C57BL/6
Sex: female
genotype/variation: double WT
age: 1 year
mouse status: Healthy
tissue/cell type: Basophilic(II) and polychromatic(III) erythroblasts
|
| Treatment protocol |
100ng/mL recombinant mouse IL-6 were added into the EPO-containing medium at the beginning of the in vitro culture.
|
| Growth protocol |
The bone marrow lineage negative/progenitor cells were purified according to the manufacturer’s instructions using the biotin mouse lineage panel (BD Pharmingen,Cat#559971). These cells were cultured with IMDM containing 15% fetal bovine serum, 1% bovine serum albumin, 10μg/ml recombinant human insulin, 200μg/ml recombinant human holo-transferrin, 0.1 mM β-mercapto-ethanol, 1% penicillin-streptomycin, 2 mM L-glutamine, 2U/µL erythropoietin for different time points.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated by Trizol, according to the manufacturer’s instructions.
|
| Label |
biotin
|
| Label protocol |
Microarray was performed at the Genomics Core Facility at the University of Chicago. The RNA integrity was examined on a bioanalyzer (Agilent). High-quality RNA (RNA integrity number >8.5) was used for expression microarray analysis. 700 pg of total RNA was used to generate double strand cDNA, which was fragmented and biotin labeled according to Affymetrix GeneChip Pico Reagent Kit manual (Thermo Fisher).
|
| |
|
| Hybridization protocol |
5.5 μg of the fragmented and labeled cDNA was hybridized to Mouse Clariom S array for 16 h at 45°C and 60 rpm in an Affymetrix Hybridization Oven 640. Arrays were washed and stained with Streptavidin Phycoerythrin in an Affymetrix Fluidics Station 450 according to Affymetrix GeneChip Pico Reagent Kit guide (Thermo Fisher).
|
| Scan protocol |
The arrays were scanned using the Affymetrix Gene Chip Scanner 3000 7G and CEL. intensity files were generated by Affymetrix GeneChip Command Console Software (AGCC, Thermo Fisher).
|
| Data processing |
The Affymetric Clariom S microarrays were further analyzed using Affymetrix Expression Console with the SST-RMA procedure. The gene-level differential expression was performed using the Affymetrix Transcriptome Anaylsis Console (TAC) Software using default parameters. The significant genes were ranked by fold-change with a cutoff of 2 and the ANOVA p-value less than 0.05 according to Affymetrix.
affymetrix-algorithm-name = sst-rma-gene-full
affymetrix-algorithm-version = 1.0
affymetrix-array-type = Clariom_S_Mouse
program-name = Expression Console
program-version = 1.4.1.46
|
| |
|
| Submission date |
May 01, 2017 |
| Last update date |
Jan 23, 2018 |
| Contact name |
Peng Ji |
| E-mail(s) |
Peng-Ji@fsm.northwestern.edu
|
| Phone |
312-503-0451
|
| Organization name |
Northwestern University
|
| Department |
Pathology
|
| Lab |
Peng Ji's Lab
|
| Street address |
303 E. Chicago Ave
|
| City |
Chicago |
| State/province |
IL |
| ZIP/Postal code |
60611 |
| Country |
USA |
| |
|
| Platform ID |
GPL23038 |
| Series (1) |
| GSE98375 |
Transcriptional alterations in mDia1-miR146a double deficient erythroblasts and IL-6 treated erythroblasts |
|
