GSM7679875: 02-mo CD45- thymic endothelial cells - day 1 after SL-TBI [singe cell]; Mus musculus; RNA-Seq
All steps were performed at 4 C unless indicated. 10-15 thymi of 2-month-old or 18-month-old female C57BL/6 mice were excised and enzymatically digested. Briefly, thymi were mechanically dissociated into ca. 2 mm pieces. Tissue pieces were incubated with a digestion buffer (RPMI, 10% FCS, 62.5 um/mL liberase TM, 0.4 mg/ml DNase I) twice for 30 min at 37 C. Between incubation steps, supernatant containing dissociated cells was transferred to 50 mL conical tubes equipped with 100 um filter. Cells were pelleted by centrifugation at 400g for 5 min. Cell pellets were incubated with anti-mouse CD45 microbeads and CD45-positive cells were depleted from cell suspension using magnetic-associated cell sorting (MACS) on LS columns according to manufacturer's protocol. Following red blood cell lysis using ACK buffer, the CD45-depleted cell fraction was incubated with an antibody cocktail for 15 min at 4 C and cells of interest were purified by fluorescent-associated cell sorting (FACS) on a BD Biosciences Aria II using a 100 um nozzle. Cells were sorted into tubes containing RPMI supplemented with 2% BSA. FACS-purified cells were spun down at 400g for 5 min and resuspended in PBS supplemented with 0.04 % BSA for generation of single-cell suspensions. The single-cell RNA-seq of FACS-sorted cell suspensions was performed on Chromium instrument (10X genomics) following the user guide manual (CG00052 Rev E) and using Single Cell 3' Reagent Kit (v2). The viability of cells prior to loading onto the encapsulation chip was 73-98%, as confirmed with 0.2% (w/v) Trypan Blue stain. Each sample, containing approximately 8000 cells, was encapsulated in microfluidic droplets at a final dilution of 66–70 cells/µl (a multiplet rate ~3.9%). Following reverse transcription step the emulsion droplets were broken, barcoded-cDNA purified with DynaBeads and amplified by 12-cycles of PCR: 98 ̊C for 180 s, 12x (98 ̊C for 15 s, 67 ̊ C for 20 s, 72 ̊C for 60 s), and 72 ̊C for 60 s. The 50 ng of PCR-amplified barcoded-cDNA was fragmented with the reagents provided in the kit, purified with SPRI beads and resulting DNA library was ligated to the sequencing adapter followed by indexing PCR: 98 ̊C for 45 s; 12x 98 ̊C for 20 s, 54 ̊C for 30 s, 72 ̊C for 20 s), and 72 ̊C for 60 s. The final DNA library was double-size purified (0.6–0.8X) with SPRI beads and sequenced on Illumina Nova-Seq platform (R1 – 26 cycles, i7 – 8 cycles, R2 – 70 cycles or higher) at depth of 200-270 million reads per sample.