Strain:CBA/Ca; Gender: female; Age: ~7 weeks; Tissue: cultured bone marrow; Culture time: 7 days; Culture medium: RPMI 1640, 10% FCS, 2 mM L-glutamine, 50 u/ml Penicillin, 50 microg/ml streptomycin, 5x10^-5M 2-ME, ~25 ng/ml recombinant GM-CSF (supplied as x-63 transfected tissue culture supernatant); Treatment: 1 microg/ml LPS added for the final 18-20 hr of culture
Biomaterial provider
Paul Fairchild, Therapeutic Immunology Group, Sir William Dunn School of Pathology
Treatment protocol
1 microg/ml LPS (E. Coli) added on day 6 for the final 18-20 hr of culture
Growth protocol
Bone marrow, isolated from the femurs and tibias of CBA/Ca mice, were made into single cell suspension by sieving through a 70 micron nylon mesh. Red cells were lysed using ammonium chloride, washed and plated in 10cm dishes in RPMI 1640, 10% FCS, 2 mM L-glutamine, 50 u/ml Penicillin, 50 microg/ml streptomycin, 5x10^-5M 2-ME, ~25 ng/ml recombinant GM-CSF (supplied as x-63 transfected tissue culture supernatant). Cells in suspension were removed and culture medium replaced on days 3 and 6. On day 6 the medium was supplemented with 1 microg/ml LPS and the cells cultured for a further 18-20 hr, when semi-adherent cells were harvested by gentle pippetting.
Extracted molecule
polyA RNA
Extraction protocol
Total RNA was prepared using an RNeasy midi kit (Qiagen), including treatment with DNase I to remove any genomic contamination and poly A+ RNA isolated using (dT)25-coated Dynabeads (Dynal) according to the respective manufacturer's protocols.
Description
Double stranded cDNA was generated from polyA+ RNA using a cDNA synthesis kit (Roche), primed using a biotinylated dT20 primer and a long SAGE library generated essentially according to the original description by Saha et al., Nature Biotechnology, (2002)20:508-512, including the use of biotinylated linkers as described by Powell, NAR (1998)26:3445-6.
Data processing
SAGEScreen software (Akmaev et al, Bioinformatics, (2004) 20:1254-63) was used to correct for non-random sequence errors within the ditags and to extract the 17 bp long SAGE tags
