title: GSM4085512: human_spleen; Homo sapiens; RNA-Seq
library_construction_protocol: Fresh spleen tissue was obtained from patients undergoing surgical resection of tumors in other organs. Patients were not receiving treatment at the time of surgery and had not received prior chemotherapy or immunotherapy. Spleen tissue was dissociated by manual mincing followed by an incubation of 45 min at 37°C, 250 rpm,  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). Cells were then washed and filtered with a 100 μm filter. Human DCs from spleen were enriched with Dynabeads™ Human DC Enrichment kit (Thermofisher). Lin(CD3, CD19, CD56)–CD14–CD11c+MHCII+ cells were then FACS-isolated into 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).