Bone marrow cells were harvested and washed in the same manner as used for BMM preparation, except that DMEM was substituted for RPMI-1640. The cells were resuspended for differentiation in DMEM+Pen/Strep+Glutamax+10% heat inactivated FCS with 50 µg/mL ascorbic acid + 20 ng/mL human soluble RANKL (Peprotech) + 1 x 104 U/mL human soluble CSF-1 (Cetus). Medium was changed on day 3 and day 6, and cells were harvested for RNA on day 7. Medium was changed on day 3 and day 6, and cells were harvested for RNA on day 7.
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated from cell pellets using the RNeasy kit (Qiagen, Valencia, CA, USA).
Label
biotin
Label protocol
5 ug total RNA was used in a standard Affymetrix single amplification protocol.
Hybridization protocol
standard Affymetrix procedures except for use of custom GNF chip washer
Scan protocol
standard Affymetrix procedures using GCS3000 scanner
