NextSeq 500 sequencing; RNA-seq of Mus musculus BM-DCs upon stimulation by lung fibroblast conditioned medium or purified PGE2
RNA-seq of Mus musculus BM-DCs upon stimulation by lung fibroblast conditioned medium or purified PGE2
Sample 7_s
Bone-marrow cells from naïve mice were isolated by flushing femurs, tibias and humeri with RPMI-1640 medium. Following suspended in ACK lysis buffer to lyse red blood cells, bone marrow cells were passed through a 40 µm filter. Then cells were resuspended and cultured in complete RPMI-1640 medium containing GM-CSF and IL-4. The cultured medium was entirely discarded on day 4 and new warmed medium supplemented with GM-CSF and IL-4 was added. On day 7, the cultured cells were collected and used for further experiments. BM-DCs were incubated with WT or Ptgs2-/- lung fibroblast-derived conditioned medium or purified PGE2. Total RNA was isolated from BM-DCs using the miRNeasy Mini kit (Qiagen). Stranded libraries were prepared using the KAPA mRNA HyperPrep Kit: isolation of polyA containing mRNA using oligo-dT magnetic beads, RNA fragmentation, first and second strand cDNA synthesis, ligation of Illumina-specific adapters containing a unique barcode sequence for each library, and PCR amplification. Libraries were checked for quality and concentration using the DNA 1000 assay and quantitative PCR, following the manufacturers' instructions.