|
| Status |
Public on Jun 14, 2016 |
| Title |
LSK cells Abi-1 KO, biological rep3 |
| Sample type |
RNA |
| |
|
| Source name |
LSK cells from bone marrow, Abi-1 KO
|
| Organism |
Mus musculus |
| Characteristics |
age: 12-14 weeks old
tissue: bone marrow
genotype: Abi-1 KO
|
| Treatment protocol |
LSK cells were sorted and frozen.
|
| Growth protocol |
8-9 weeks old Abi1(fl/fl);Tg (Mx1- cre(-)) or Abi1(fl/fl);Tg (Mx1-cre(+)) mice were subjected to polyinosinic:polycytidylic acid [poly(I:C)]-induced activation (3 injections, each on every other day within the one week period) of the Cre recombinase under control of the Mx1 promoter to obtain animals with an Abi1(fl/fl);Tg (Mx1-cre(-)) (Abi-1 WT) or Abi1(-/-);Tg (Mx1-cre(+)) (Abi-1 KO) genotype. Recombination and Abi-1 gene inactivation was confirmed in the bone marrow of 12-14 weeks old mice. LSK cells were obtained from the bone marrow samples, in which the recombination was confirmed by PCR.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
To isolate LSKs flushing method was used. For sorting LSKs or for frequency analysis, lineage-depleted bone marrow cells were stained with BUV395 Streptavidin, APC CD117/c-Kit (2B8), and PE.Cy7 Ly-6A/E/Sca-1 (D7) (BD Pharmingen, San Diego,CA). Total mRNA from LSK cells was purified using a PureLink RNA Mini Kit from Ambion/Life Technologies (Grand Island, NY). The Affymetrix WT Pico Expression Kit (Affymetrix, INC., Louisville, KY) was used according to the manufacturer’s instructions. The quality of the input RNA was assessed using an Agilent Bioanalyzer Picochip. Preamplification was performed on 5 ng total RNA followed by the initial reverse transcription reaction. The cDNA was converted to dsDNA, which was then amplified and converted in an in vitro transcription reaction, yielding between 25 and 75 μg purified cRNA. A 20 μg sample of cRNA was converted to 2nd cycle cDNA.
|
| Label |
biotin
|
| Label protocol |
Samples were enzymatically fragmented and biotinylated using the WT Terminal Labeling Kit (Affymetrix)
|
| |
|
| Hybridization protocol |
After purification, a 5.5 μg sscDNA sample was fragmented, and a 3.22 μg sample was end-labeled and hybridized to mouse Gene ST arrays overnight at 45°C, 60 rpm. The wash/stain reaction was carried out on a FS450 Fluidics Station using Fluidics protocol FS450-0001.
|
| Scan protocol |
The arrays were scanned on an Affymetrix 3000 7G gene scanner.
|
| Description |
LSK-enriched population of hematopoietic progenitor cells isolated via sorting from the whole bone marrow of Abi-1 KO mouse
|
| Data processing |
Partek Genomics Suite version 6.6 software was used for quality control analysis and determination of differentially expressed genes. Data were analyzed using ANOVA with repeated measures analysis. Changes of 1.5 fold above or below baseline with FDR corrected p-values of ≤0.05 were considered significant.
MTA-1_0.r3.pgf
MTA-1_0.r3.Psrs.mps
|
| |
|
| Submission date |
Jun 13, 2016 |
| Last update date |
Jun 14, 2016 |
| Contact name |
Patrycja M Dubielecka |
| E-mail(s) |
[email protected]
|
| Phone |
401-444-8391
|
| Organization name |
Rhode Island Hospital and Warren Alpert Medical School at Brown University
|
| Department |
Medicine/Hem-Onc
|
| Lab |
Signal Transduction Lab
|
| Street address |
One Hoppin street
|
| City |
Providence |
| State/province |
RI |
| ZIP/Postal code |
02903 |
| Country |
USA |
| |
|
| Platform ID |
GPL20775 |
| Series (1) |
| GSE83288 |
Expression profiling of Abelson interactor-1 (Abi-1) deficient hematopoietic progenitor cells. |
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