Stimulation of cells was carried out for up to 48 hours in six well plates with 5,000,000 cells/well in a total of 5 ml medium, set up immediately prior to addition of 1 μg/ml LPS (Sigma: L2630).
Growth protocol
BL20, an uninfected bovine lymphosarcoma cell line (Olobo and Black, 1989. Vet. Immunol. Immunopathol. 20, 165-172) and TBL20, a T. annulata (Ankara strain) infected BL20 cell line (Shiels et al., 1986. Vet. Parasitol. 21, 1-10.) were cultured in standard complete medium (Swan et al., 1999. Mol. Biochem. Parasitol. 101, 117-129) except that heat-inactivated foetal bovine serum (FBS; Sigma) was used. Cultures were seeded with 200,000 cells/ml and maintained by feeding with fresh medium every two to three days (Schmuckli-Maurer et al., 2010. Cell Microbiol. 12, 158-173).
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted using Triazol (ref) and this was cleaned up using an RNeasy Mini kit (Qiagen Inc., CA, USA) . RNA quality and concentration was determined by analysis with an Agilent 2100 bioanalyzer (ref)
Label
Cy3
Label protocol
Labeling was performed by NimbleGen Systems Inc., Madison, WI USA, following their standard operating protocol. See www.nimblegen.com.
Hybridization protocol
Hybridization was performed by NimbleGen Systems Inc., Madison, WI, USA following their standard operating protocol. See www.nimblegen.com.
Scan protocol
Scanning was performed by NimbleGen Systems Inc., Madison, WI USA, following their standard operating protocol. See www.nimblegen.com.
Description
This sample is of the bovine lymphosarcoma cell line BL20, 4 hours post LPS stimulation, this is the 3rd of 3 replicates
Data processing
The raw data (.pair file) was subjected to RMA (Robust Multi-Array Analysis; Irizarry et al. Biostatistics 4(2):249), quantile normalization (Bolstad et al. Bioinformatics 19(2):185), and background correction as implemented in the NimbleScan software package, version 2.4.27 (Roche NimbleGen, Inc.).
Submission date
Mar 12, 2012
Last update date
Oct 01, 2012
Contact name
William Weir
Organization name
University of Glasgow
Department
Institute of Infection, Immunity and Inflammation
Lab
Theileria lab
Street address
Room 220, Henry Wellcome Building, Garscube Campus, Bearsden Road
