title: GSM8442461: PolyA enriched RNAseq from cDC2; Mus musculus; RNA-Seq
library_construction_protocol: Cells were sorted with the Flow cytometry (FC) before sequencing. Organs were smashed into small pieces and then incubated 15min at 37°C in agitation with a tissue digestion solution composed of 1 mL IMDM (Corning) supplemented with 0.35 mg/mL collagenase type IV (Sigma-Aldrich/ SCR103), 1 mg/ml dispase II (Gibco/ 17105041) and 0.2 mg/ml DNAse (Roche/ 11284932001). The tissue was then further dissociated by pipetting and filtered using 40 μm cell strainers (Corning/ 352340). Single cell solution was washed with 30 mL of autoMACS Running Buffer (Miltenyi Biotec/130-940 091-221) and pelleted for 5min 300g 4°C to remove excess enzymes. Samples were transferred to FACS tubes, stained with surface antibody. Cells were sorted according to GFP and cell marker positivity: B cells (CD45+CD19+), RPM (CD45+F4/80+), cDC1(CD45+CD11c+CD8a+), cDC2(CD45+CD11c+Cd11b+), LSEC(CD45-CD31+), KC(CD45+,F4/80+). Cells were sorted from nr=3 animals per pool, adding up to 500k-3mln cells per sample. After sorting, cells were pelleted 300g for 5min 4°C, resuspended in 700uL QIAzol buffer and total RNA was extracted with miRNeasy Mini kit(QIAgen/ 217004) following manifacturer instruction. Libraries were prepared using QIAseq UPX 3' Transcriptome Kit (QIAgen/ 333068) following manifacturer instruction.