title: GSM4085510: Tbx21CreRFPpos; Mus musculus; RNA-Seq
library_construction_protocol: Spleens were digested in collagenase in RPMI1640 supplemented with 5% fetal calf serum, 1% L-glutamine, 1% penicillin–streptomycin, 10 mM HEPES, 1 mg/ml collagenase A (Sigma, 11088793001) and 1U/mL DNase I (Sigma, 10104159001) for 45 min at 37°C, 250 rpm. Samples were washed and incubated in digest solution for 30 min. Digested samples were filtered through 100-μm strainers and centrifuged to remove collagenase solution. Mouse DC subsets were enriched from a pool of two spleens with Dynabeads™ Mouse DC Enrichment Kit (Invitrogen). Lin(CD3, CD19, CD90)–CD64–Ly6C–CD11c+MHCII+ cells were then purified using an Aria II cell sorter (BD Bioscience) into two populations (RFP+ and RFP– cells). Cells were sorted into cRPMI, before being pelleted and resuspended in RPMI-2% FBS for single-cell RNA seq. The scRNA-seq libraries were prepared following the user guide manual (CG00052 Rev E) provided by the 10X Genomics and Chromium™ Single Cell 3' Reagent Kit (v2). Briefly, samples were encapsulated in microfluidic droplets at a dilution of ~70 cells/µl (doublet rate ~3.9%.). Encapsulated cells were subjected to reverse transcription (RT) reaction at 53ºC for 60 min. After RT step, the emulsion droplets were broken and barcoded-cDNA was purified with DynaBeads, followed by 14-cycles of PCR-amplification (98 °C for 180 s; [98 °C for 15 s, 67 °C for 20 s, 72 °C for 60 s] x 12-cycles; 72 °C for 60 s). 50 ng of PCR-amplified barcoded-cDNA was fragmented with the reagents provided in the kit and purified with SPRI beads to obtain an average fragment size of 600 bp. Next, the DNA library was ligated to the sequencing adapter followed by indexing PCR (98 °C for 45 s; [98 °C for 20 s, 54 °C for 30 s, 72 °C for 20 s] x 10 cycles; 72 °C for 60 s). The resulting DNA library was double-size purified (0.6-0.8X) with SPRI beads and sequenced on an Illumina NovaSeq platform  (R1 – 26 cycles, i7 – 8 cycles, R2 – 96 cycles) resulting in 184.5-186.1 million reads per sample (average reads per single-cell being 42,000 and average reads per transcript 4.40-7.14).