GSM3896274: Spleen CD8+ dendritic cells, replicate 2; Mus musculus; RNA-Seq
Single cell suspensions were obtained by enzymatic digestion with 2.5 mg/ml collagenase D (Roche via Sigma Aldrich) of the applicable organ from different cohorts of naïve C57Bl/6 mice (NCI/Charles River). Whole digests were magnetically enriched with Miltenyi microbeads for cells of interest before FACS sorting to purify (BD FACS Aria Fusion). Resulting cells were lysed with RLT buffer and total RNA was extracted using the Qiagen RNeasy micro or mini kits. A modified Clontech SMARTer Ultra Low Input RNA Kit for Sequencing - v3 (Mountain View, CA, USA) and modified Kapa Biosystems (Wilmington, MA, USA) KAPA Hyper Prep Kit was used to primarily target all polyA RNA. Library construction started from isolation of total RNA species, followed by mRNA (poly-A) isolation, 1st and 2nd strand cDNA synthesis, adapter ligation, amplification and bead templating.