title: Single cell RNA sequencing of thymic dendritic cells reveals molecular determinants of antigen transfer in the thymus
design: Single cell RNA sequencing of thymic dendritic cells reveals molecular determinants of antigen transfer in the thymus
library_construction_protocol: Mice were euthanized by cervical dislocation. Thymi were excavated using forceps, cut into 10-15 pieces and digested by enzymatic cocktail of 0.1 mg/ml Collagenase D (Roche) and DNAse I (40 U/ml; Roche) dissolved in RPMI medium supplemented with 3% FBS. Note that pieces of each thymus were put into 1 ml of enzymatic cocktail in 1.5 ml eppendorf tube. To isolate thymic epithelial cells, 0.1 mg/ml Dispase II (Gibco) was added to the enzymatic cocktail. To complete digestion, enzymatic cocktails with thymi were put into thermoshaker, which was set to 37 oC and 800 rpm, and incubated for ~80 minutes. After the incubation, remaining non-digested thymic pieces were pipetted up and down several times using cut tip and the whole solution was transferred into 15 ml falcon tube through filter. Each thymic solution was washed with 2 ml of ice cold 3% FBS and 2 mM EDTA solution in PBS to stop the enzymatic reaction. Then, thymi were spun down (4°C, 400 x g, 10 min). mRNA was extracted from cell suspension during library preparation routine using Chromium next gem single-cell 3′ reagent kit (version 3.1). Cells were resuspended with cocktail of anti-CD11C and anti-CD11B antibodies both conjugated with biotin, stained on ice for 25 min, washed with 3 % FBS and 2 mM EDTA solution in PBS and then spun down (4°C, 300 x g, 10 min). Next, cells were stained with anti-biotin magnetic beads (Miltenyi) according to manufacturer´s protocol and CD11C+ and CD11B+ cells were MACS-enriched using QuadroMACS (Miltenyi). Enriched cells were stained with fluorochrome-conjugated streptavidin on ice for 15 min, washed with 3 % FBS and 2 mM EDTA solution in PBS and then spun down (4°C, 300 x g, 10 min). Next, ~100000 of Streptavidin+ cells were sorted using cell sorter BD Influx (BD Biosciences) from a pool of three littermate thymi. Importantly, half of the cells was sorted as TdTOMATO+ (CAT-experienced cells) and the other half as TdTOMATO– (CAT inexperienced cells) into separate collection tubes to prepare two distinct scRNAseq libraries (samples). To check that viability of sorted cells is higher than 90%, an automated TC20 cell counter (Bio-Rad) was used. ScRNAseq libraries were prepared using Chromium controller instrument and Chromium Next Gen single-cell 3' reagent kit version 3.1 (both 10X Genomics) according to the manufacturer´s protocol targeting 4000 cells per sample. Thus, note that we sequenced 4000 of TdTOMATO+ as well as TdTOMATO– cells.