primary host: W41/W41 secondary host strain: C57BL/6 gender of the clone: male strain of the clone: C57BL/6-GFP age of the clone: 29 months cell type: lymphoid-biased (Ly-bi) hematopoietic stem cells
Extracted molecule
total RNA
Extraction protocol
Bone marrow from clonally repopulated hosts was harvested and donor type HSC were enriched for Donor type+, Sca-1+, and Lin- cells by flow cytometry. Whole RNA was isolated from about 5x10^4 cells using RNeasy® (Qiagen, Valencia,CA, USA) and was linearly amplified as described (Bystrykh et. al., 2005, #391).
Label
Cy3
Label protocol
To generate Cy3-labeled cDNA probes, aRNA (5ug) and random hexamers (5ug, Pharmacia) were mixed in a total volume of 26 ul (containing RNase-free H2O), heated to 70 °C for 10 min and chilled on ice. Then, 10ul 5X first-strand buffer (Life Technologies), 5ul 0.1 M DTT (Life Technologies), 1.5ul Rnase Block (Stratagene), 1ul 25 mM d(GAT)TP and 2ul 1mM dCTP (Pharmacia), 2ul Cy3-dCTP (Amersham) and 2.5ul Superscript RT II (Life Technologies) were added and incubated at room temperature for 10 min and then at 37 °C for 2 h. To degrade the aRNA template, 1ul RNaseA (Amersham) and 5ul RNaseH (Life Technologies) was added and incubated at 37 °C for 20 min. The probes were purified with a Qiagen 96 PCR Purification kit. The probes were normalized by fluorescence, vacuum-dried and resuspended in 55ul of hybridization buffer containing 25% 4X hybridization buffer (Amersham), 50% formamide and 10% mouse Cot1 DNA (Life Technologies).
Hybridization protocol
Cy3-labeled probes were heated to 99°C for 5 min, room temperature for 5 min and applied to the slides. The slides were covered with glass coverslips, sealed with DPX (Fluka, Milwaukee, Wisconsin) and hybridized at 42 °C for 14–16 h. The slides were washed in 1X SSC, 0.2% SDS at 55 °C for 5 min, 0.1X SSC, 0.2% SDS at 55 °C for 5 min. After a quick rinse in 0.1X SSC, the slides were centrifugated briefly to dry.
Scan protocol
The arrays were scanned for Cy3 fluorescence using an Array Scanner (Molecular Dynamics) and analyzed using Autogene 2.5 software (BioDiscovery, Los Angeles, California).
Data processing
The Affymetrix proprietary probes (i.e. labeled with an AFFY prefix and the GNF probes by the prefix gnf1m) were filtered out prior to any analyses. The resulting reduced data set (of 23189 ID_REF) was normalized and scaled using GCRMA (matchprobes_1.14.0 with R v.2.8.0 (2008-10-20)).
