GSM7429781: Ctrl CD8+; Mus musculus; RNA-Seq
Kidneys were minced and incubated in collagenase D solution for 30 minutes at 37°C. Single-cell suspensions of kidney digestions were obtained by mechanical disruption of tissues using 70-μm strainers and KMNC were isolated using Percoll density gradient centrifugation. The numbers of viable lymphocytes in each sample were determined using trypan blue exclusion dye under a light microscope.   T lymphocyte cell staining was performed using standard methods. Briefly, cells were stained in FACS buffer (PBS, 2% FBS, 0.1% sodium azide) and preincubated with anti-CD16/CD32 antibody for 10 minutes to minimize nonspecific binding through Fc-receptors. We used the following mAbs: mCD45–APC- Cyanine7 (30-F11), mTCR- BV421 (H57-597), mCD8α-APC (53–6.7), mCD4- PerCP-Cyanine5.5 (GK1.5), and live/ dead fixable aqua dead cell stain kit for 405 nm excitation.   Cells were stained by incubation with the appropriate cocktails of fluorochrome-conjugated monoclonal antibodies for 30 min at 4°C, washed, and stained with live/ dead aqua followed by incubation for 30 minutes. Cells were then sorted with a FACS Aria II Cell Sorter. After sorting purified CD4+, CD8+, and DN T cells from normal and ischemic mice were used for single cell RNA-Seq analysis. To capture and barcode individual cells using 10X Chromium technology, purified sorted cells were processed for high-throughput droplet encapsulation. Cell counts and viability were determined using the Cell Countess II with Trypan Blue.  A maximum volume of 43.3 uL/sample was used for processing to target up to 10,000 cells.  Cells were combined with RT reagents and loaded onto 10X Next GEM Chip G along with 3' v3.1 gel bead. The NextGEM protocol was run on the 10X Chromium Controller to create GEMs, composed of a single cell, uniquely barcoded gel bead, and RT reagents.  100uL of emulsion was retrieved from the chip and incubated (45 min at 53°C, 5 min at 85°C, cool to 4°C), generating barcoded cDNA from each cell.  The GEMs were broken using Recovery Agent and cDNA was cleaned, following manufacturer's instructions using MyOne SILANE beads.  cDNA was amplified for 11-12 cycles, depending on input cell number (3 min at 98°C, 11-12 cycle: 15sec at 98°C, 20sec at 63°C, 1min at 72°C; 1min at 72°C, cool to 4°C).  Samples were cleaned using 0.6X SPRIselect beads. QC was completed using Qubit and Bioanalyzer to determine size and concentrations.  10uL of amplified cDNA was carried into library prep.  Fragmentation, end repair and A-tailing were completed (5 min at 32°C, 30min at 65°C, cool to 4°C), and samples cleaned up using double sided size selection (0.6X, 0.8X) with SPRIselect beads.  Adaptor ligation (15min at 20°C, cool to 4°C), 0.8X cleanup and amplification were performed, with PCR using unique i7 index sequences.  Libraries underwent a final cleanup using double sided size selection (0.6X, 0.8X) with SPRIselect beads.  Library QC was performed using Qubit, Bioanalyzer and KAPA library quantification qPCR kit.  Libraries were sequenced on the Illumina NovaSeq 6000 using v1.5 kits, targeting 50K reads/cell, at read lengths of 28 (R1), 8 (i7), 91 (R2).  Demultiplexing and FASTQ generation was completed using Illumina's BaseSpace software.