|
| Status |
Public on Oct 21, 2016 |
| Title |
g5 2 |
| Sample type |
RNA |
| |
|
| Source name |
BM LSK SLAM, G-CSF, 5 days (9 doses) mouse 2
|
| Organism |
Mus musculus |
| Characteristics |
tissue: bone marrow
gender: male
treatment: G-CSF (100 ug/kg/injection, q12h, 5 days
|
| Treatment protocol |
HSCs from control mice or after 2 or 5 days of G-CSF treatment or 14 days after POL5551 infusion start
|
| Growth protocol |
HSCs harvested directly from mouse by flow cytometry
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was prepared using the NucleoSpin® RNA XS Isolation kit (Macherey-Nagel, Bethlehem, PA, USA) following the manufacturer's recommendations. The protocol includes an on-column DNase digestion. RNA was quantified using a NanoDrop-1000 spectrophotometer and quality was monitored with the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).
|
| Label |
Cy5
|
| Label protocol |
cDNAs were chemically labeled with Kreatech ULS RNA labeling kit (Kreatech Diagnostics). Per reaction, 3ug of each RNA (+water=16ul) was mixed with Kreatech 10x labeling buffer (2ul) and Kreatech cy5/DY-ULS (2ul). The reactions were incubated at 85C for 15 minutes in the dark and placed on ice for 3 minutes. Labeled cDNA was purified with Qiagen PCR purification columns according to manufacturer’s protocol. cDNAs were quantitated on a Nanodrop spectrophotometer. Detailed protocol can be found at http://www.kreatech.com/fileadmin/user_upload/Documenten/PDF/12_man_EA-021-022-023__D0.6.pdf
|
| |
|
| Hybridization protocol |
The balanced aRNAs were suspended in Agilent 2X Gene Expression buffer (55ul), Agilent 10X Blocking agent (11ul), and Kreablock (6ul). The hybridization solutions were applied to Agilent Mouse v2 8x60K microarrays (G4852B-074809). Hybridization was carried out at 65C for 20 hours. Washing procedures were carried out according to Agilent gene expression protocols.
|
| Scan protocol |
Slides were scanned on an Agilent C-class Microarray scanner to detect Cy5 fluorescence, according to manufacturer's specifications.
|
| Data processing |
Gridding and analysis of images was performed using Feature Extraction (v11.5.1.1, Agilent Technologies).
|
| |
|
| Submission date |
Oct 20, 2016 |
| Last update date |
Oct 21, 2016 |
| Contact name |
Darja Karpova |
| E-mail(s) |
d.karpova@wustl.edu
|
| Phone |
+1-314-362-9335
|
| Organization name |
Washington University School of Medicine
|
| Department |
Oncology, BMT
|
| Lab |
John F. DiPersio
|
| Street address |
660 S Euclid Ave
|
| City |
St. Louis |
| State/province |
MO |
| ZIP/Postal code |
63110 |
| Country |
USA |
| |
|
| Platform ID |
GPL21163 |
| Series (1) |
| GSE88999 |
Gene expression profiling of differentially treated hematopoietic stem cells (LSK SLAM) |
|
