GSM5548114: DC BM2; Mus musculus; RNA-Seq
Isolation of DCs from spleen or bone marrow (BM) from 8-12 wk old C57BL6 mice; for BM the femurs, tibia, pelvis, humerus and sternum was used. For each sort the DCs were enriched with CD11c microbeads from 2 mice per sample. These DC-enriched samples were sorted for CD11c+MHCII+CD11bhighCD24low cDC2 RNA was extracted with RNeasy Micro kit (Qiagen) according to the manufacturers protocol. RNA quality check, polyA RNA selection and sequencing were performed at GenomeScan BV (Leiden, The Netherlands). RNA quality and quantity was determined by a Bioanalyzer Picochip (Agilent). The NEBNext Ultra Directional RNA Library Prep Kit for Illumina was used to process the sample(s). The sample preparation was performed according to the protocol "NEBNext Ultra Directional RNA Library Prep Kit for Illumina" (NEB #E7420S/L). Briefly, mRNA was isolated from total RNA using the oligo-dT magnetic beads. After fragmentation of the mRNA, a cDNA synthesis was performed. This was used for ligation with the sequencing adapters and PCR amplicification of the resulting product. The quality and yield after sample preparation was measured with the Fragment Analyzer. The size of the resulting products was consistent with the expected size distribution (a broad peak between 300-500 bp). Per sample, 25 ng RNA was used for library preparation and single-end, 75 nucleotide fragments were analyzed by Illumina NextSeq 500. A concentration of 1.6 pM of DNA was used.