GSM7823615: 02mo CD45+ RAG2GFP- thymic cells - steady state biological rep2 [single 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 RAG2GFP.C57BL/6 mice were injected with 3 μg of conjugated CD45 antibody and sacrificed 3 min later. The thymic tissue was 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. The fraction of CD45+ peripheral i.v.-labeled cells where removed and cells where sorted for RAG2-GFP- cells after removal of  TER119 and DAPI+ cells. Cells were sorted into tubes containing RPMI supplemented with 2% BSA. FACS-purified cells were spun down at 1200 RPM for 5 min and resuspended in PBS supplemented with 0.04 % BSA for generation of single-cell suspensions. Viability of cells was confirmed to be above 80%. 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.