GSM8589625: pDC, bone marrow, sorted from naïve mice BM, rep.4; Mus musculus; RNA-Seq
To prepare single cell lung suspensions for flow cytometry. After perfusing murine lungs, they were placed in a gentle MACS™ C tube and mechanically homogenized in 5 ml RPMI-1640, 10% FBS, and 0.1 mg/ml DNAse using a gentle MACS™ Octo Dissociator (Miltenyi Biotech) without the use of collagenase. The lung cell suspensions were then lysed of RBCs, enumerated, and stained with fluorophore-conjugated antibodies prior to flow cytometric analysis. We used BD Aria for flow cytometric sorting, cell always stays on ice or sorted at 4℃. Neutrophils were identified as CD45+ CD11b+ Ly6Clo Ly6G+ cells, pDCs as CD45+ CD11cint SiglecF- CD19- NK1.1- CD11b- B220+ SiglecH+ cells After RiboGreen quantification and quality control by Agilent BioAnalyzer, 1.9-2.0 ng total RNA with RNA integrity numbers ranging from 7.8 to 10 underwent amplification using the SMART-Seq v4 Ultra Low Input RNA Kit (Clonetech cat. no.  63488), with 12 cycles of amplification. Subsequently, 7.4-10 ng of amplified cDNA was used to prepare libraries with the KAPA Hyper Prep Kit (Kapa Biosystems cat. no. KK8504) using 8 cycles of PCR. Samples were barcoded and run on a HiSeq 4000 or NovaSeq 6000 in a PE50 (HiSeq) or PE100 (NovaSeq) run, using the HiSeq 3000/4000 SBS Kit or NovaSeq 6000 S4 Reagent Kit (200 Cycles) (Illumina).  An average of 40 million paired reads were generated per sample and the percent of mRNA bases per sample averaged 84%.