title: GSM5820989: scRNA+TCR-seq of tdTomato+ GFP+ CD4 T cells from the colonic lamina propria [TCR]; Mus musculus; RNA-Seq
library_construction_protocol: For isolation of CD4 T cells from the colon, tissues were enzymatically digested with DTT/EDTA buffer (PBS with 2mM L-glutamine, 10mM HEPES, 1x penicillin/streptomycin, 5% FCS, 1mM DTT and 2mM EDTA) for 15 minutes for 20 minutes at 37°C shaking at 250 RPM. Cells were filtered through a 100-µm nylon mesh to collect the intraepithelial fraction. Remaining colon tissue was further digested with Collagenase/DNAse buffer (RPMI-1640 with 2mM L-glutamine, 10mM HEPES, 1x penicillin/streptomycin, 5% FCS, 1 mg/ml collagenase A, and 1 µg/ml DNaseI) for 25 minutes at 37°C shaking at 250 RPM in the presence of five 0.25-inch ceramic spheres. Cells were filtered through a 100-µm nylon mesh to collect the lamina propria fraction. Cells were stained using fluorescent dye-conjugated antibodies and tdTomato+GFP+ and tdTomato-GFP- colonic lamina propria CD4 T cells were isolated using BD Aria II sorter. Single-cell RNA-Seq of FACS-sorted cell populations was performed on a Chromium instrument (10X Genomics) following the user guide manual for 3′ v3 or 5′ v2 chemistry. In brief, FACS-sorted cells were washed once with PBS containing 0.04% bovine serum albumin (BSA) and resuspended in PBS containing 0.04% BSA to a final concentration of 700-1,200 cells per μl. The viability of cells was above 80%, as confirmed with 0.2% (w/v) Trypan Blue staining (Countess II). Cells were captured in droplets. Following reverse transcription and cell barcoding in droplets, emulsions were broken and cDNA was purified using Dynabeads MyOne SILANE (ThermoFisher: Cat # 37002D) followed by PCR amplification per manual instruction. Approximately 5,000 cells were targeted for each sample, and triplicates from the same biological group were multiplexed together on one lane of 10X Chromium to reach 15,000 cells targeted, following cell hashing with TotalSeq-C reagents. ScRNA-seq and scTCR-seq libraries were prepared using the 10x Single Cell Immune Profiling Solution Kit, according to the manufacturer's instructions. Briefly, amplified cDNA was used for both 5′ gene expression library construction and TCR enrichment. For gene expression library construction, amplified cDNA was fragmented and end-repaired, double-sided size-selected with SPRIselect beads, PCR-amplified with sample indexing primers (98 °C for 45 s; 14–16 cycles of 98 °C for 20 s, 54 °C for 30 s, 72 °C for 20 s; 72 °C for 1 min), and double-sided size-selected with SPRIselect beads. For TCR library construction, TCR transcripts were enriched from 2 μl of amplified cDNA by PCR (primer sets 1 and 2: 98 °C for 45 s; 10 cycles of 98 °C for 20 s, 67 °C for 30 s, 72 °C for 1 min; 72 °C for 1 min). Following TCR enrichment, enriched PCR product was fragmented and end-repaired, size-selected with SPRIselect beads, PCR-amplified with sample-indexing primers (98 °C for 45 s; 9 cycles of 98 °C for 20 s, 54 °C for 30 s, 72 °C for 20 s; 72 °C for 1 min), and size-selected with SPRIselect beads. Final libraries (GEX, TCR and HTO) were sequenced on Illumina NovaSeq 6000 platform (R1 – 26 cycles, i7 – 10 cycles, i5 – 10 cycles, R2 – 90 cycles).