GSM3832751: gfppos_dc1_1; Mus musculus; RNA-Seq
10e4 DC were sorted as described above into 700 mL Trizol. RNA was isolated using RNeasy Micro Kits (Qiagen). 0.5-1 ng of RNA was synthesized into cDNA using the Smart-Seq v4 Ultra Low Input RNA Kit for Sequencing (Takara Bio). Sequencing libraries were prepared using the Low Input Library Prep Kit (Takara Bio). Single cell suspensions were obtained from lung and LN by digestion with collagenase IV 0.25mg/ml (Sigma) at 37C for 30min (lung) or 25min (LN) followed by passing through a 70um cell strainer and RBC lysis (RBC lysis buffer, BioLegend) for 2min at room temperature. For flow cytometry or FACS cells were stained in FACS buffer (PBS supplemented with 2% BSA and 5mM EDTA) with mAbs specific to CD45 (clone 30-F11, BioLegend), Siglec F (clone E50-2440, BD Pharmingen), CD11c (clone N418, Invitrogen), CD24 (clone M1/69, Invitrogen), CD103 (clone 2E7, BioLegend), I-A/I-E (clone M5/114.15.2, eBioscience), CD11b (clone M1/70, eBioscience)