GSM9154332: BilateralTCR1_L003; Mus musculus; RNA-Seq
Tumors were cut into small fragments and digested for 45 minutes at 37°C with 0.2% collagenase (FUJIFILM Wako) and DNase I (Sigma-Aldrich), except for experiments for single-cell analysis, in which tumors were digested with 0.2% collagenase NB8 (Nordmark Pharma GmbH). The cells were then subjected to two-phase density separation with upper 35% Percoll PLUS (GE Healthcare) (1000g, 10 minutes at room temperature). Leukocytes were recovered, washed and resuspended in preparation medium (PM) [5%FBS, 10mM HEPES (Nacalai), Penicillin-Streptomycin (Nacalai) in RPMI-1640 (Nacalai)]. After RBC lysis with ACK buffer [155mM of NH4Cl (Nacalai), 10mM of KHCO3 (Nacalai), and 0.5mM EDTA pH8.0 (NIPPON GENE)] for 1 minute at room temperature, cells were washed and resuspended in PM, and the cell concentrations were measured using Flow-Count fluorospheres (Beckman Coulter) and a CytoFLEX S flow cytometer (Beckman Coulter).Cells were stained with a mix of Fc Block (anti-mouse CD16/CD32 mAb; clone 2.4G2, BioLegend) and fluorophore-conjugated anti-mouse monoclonal antibodies (Supplementary Table 5). Antibody mixtures were prepared using BD Horizon Brilliant Stain Buffer Plus (BD) diluted 5x with FACS buffer (D-PBS(-) supplemented with 2% FBS, and 0.05% Sodium Azide). Staining was performed for 20 minutes at 4°C. In the single-cell RNA-seq/TCR-seq experiment, cells were stained with antibodies and anti-CD45 Sample Tag oligonucleotide-conjugated antibodies from the Single-Cell Multiplexing Kit (BD Biosciences). Before sorting, T cells were enriched from tumor suspensions by further staining with BD IMag APC Magnetic Particles-DM (BD Biosciences), then placed on magnetic plate. After washing by MACS buffer (D-PBS(-) supplemented with 10% BSA, and 2 mM EDTA pH8.0), APC and APC-Cy7(CD8, mouse TCRb)-positive cells were collected as positive fraction. Cell sorting was performed using a FACS Aria II or Aria III (BD Biosciences). Cells were stained with 5µg/mL of propidium iodide (PI, Sigma-Aldrich) immediately before sorting. Nonviable cells were excluded from the analysis based on forward and side scatter profiles and PI staining. For bulk TCR repertoire analysis and bulk RNA-seq analysis, cells were sorted into Lysis buffer [1% w/v LiDS, 100 mM Tris-HCl pH7.5, 500 mM LiCl, 5 mM dithiothreitol, and 10 mM EDTA pH8.0] and stored at −80°C. For single-cell RNA/TCR-seq, cells were sorted into D-PBS(-) with 10% FBS. For the bulk TCR-seq experiments, cells were sorted from the CD8+, CD8+PD-1+Ly108+TIM-3-, CD8+PD-1+Ly108+TIM-3+, CD8+PD-1+Ly108-TIM-3+ and CD8+PD-1- fractions to produce five datasets per mouse. For the single-cell RNA/TCR-seq and bulk TCR-seq experiment, cells were sorted from the CD8+PD-1+Ly108+TIM-3-, CD8+PD-1+Ly108+TIM-3+, CD8+PD-1+Ly108-TIM-3+ and CD8+PD-1- fractions. Bulk TCR libraries were prepared on purified T cells lysed in lysis buffer [1% LiDS, 100 mM Tris-HCl (pH 7.5), 500 mM LiCl, and 10 mM EDTA]. PolyA RNAs were isolated according to a previous report with some modifications (GSE110711).To perform reverse transcription and template-swiching, beads were then suspended in 10 µL of RT mix [1× First Strand buffer (Thermo Fisher Scientific), 1 mM dNTP, 2.5 mM DTT (Thermo Fisher Scientific), 1 M betaine (Sigma-Aldrich), 9 mM MgCl2, 1 U/µL RNaseIn Plus Rnase Inhibitor (Promega), 10 U/µL Superscript II (Thermo Fisher Scientific), and 1µM of i5-TSO], and incubated for 60 min at 42ºC and immediately cooled on ice. Beads were washed once with with B&W-T buffer [5 mM Tris-HCl (pH 7.5), 1 M NaCl, 0.5 mM EDTA, and 0.1% Tween-20], and once with Tris-HCl (pH 8.0). To amplify the TCR cDNA containing complementarity determining region 3 (CDR3), nested PCR of the TCR locus was performed as follows. Beads with containing cDNA was resuspended with the 25 µL of first PCR mixture [0.4 µM of primers (i5, Trac_ex, and Trbc_ex), and 1x KAPA Hifi Hotstart ReadyMix (KAPA Biosystems, Wilmington, MA, #KK2602)], and the thermal cycling was performed as following condition: denaturation at 95°C for 3 min, 10 cycles of denaturation for 20 sec at 98°C, annealing for 15 sec at 65°C and extension for 30 sec at 72°C, followed by a final extension at 72°C for 2 min. Then, 2.5 µL of first PCR product was mixed with the 22.5 µl of second PCR mixture [0.35 µM of primers (i5_2nd, i7-BC_mTrac, and i7-BC_mTrbc), and 1x KAPA Hifi Hotstart ReadyMix], , and the thermal cycling was performed as following condition: denaturation at 95°C for 3 min, 18 cycles of denaturation for 20 sec at 98°C, annealing for 15 sec at 65°C and extension for 30 sec at 72°C, followed by a final extension at 72°C for 2 min.. The second-PCR products were purified by an Agencort AM Pure XP kit (Bexkman-Coulter, CA, #A63881) at a 0.7:1 ratio of beads to sample, and eluted with 20 µL of Tris-HCl (pH 8.0).To amplify TCR library and add adaptor sequence for next generation sequencer, the third PCR was performed as follows. 5µL of purified second PCR product was mixed with the 20 µl of third PCR mixture [0.4 µM of primers (i5-BC and i7-BC), and 1x KAPA Hifi Hotstart ReadyMix (KAPA Biosystems, Wilmington, MA, USA, #KK2602)], and the thermal cycling was performed under the same condition as first PCR, excepted for the number of cycles (23 cycles). The third-PCR products were purified as second PCR. The products were pooled and then purified and subjected to dual size selection using ProNex size-selective purification system (Promega) and eluted with 25 μL of Tris-HCl (pH 8.5). Final TCR libraries, whose lengths were about 600 base pairs were sequenced using an Illumina Novaseq 6000 S4 flowcell (67 bp read 1 and 140 bp read 2) (Illumina, USA). Only read2 contained the sequence regarding the definition of T cell clones. For the single cell library, live cells were stained with Calcein AM (Nacalai) and loaded on an BD Rhapsody cartridge (BD) and processed using BD Rhapsody Cartridge Reagent Kit (BD) following the manufacturer's instructions. Single-cell cDNA synthesis was performed using BD Rhapsody cDNA Kit (BD) following the manufacturer's protocol. Whole transcriptome analysis (WTA), TCRseq, and Sample tag Index libraries were prepared using Immune Response Panel Mm (BD) according to the manufacturer's instructions. Amplified libraries were quantified using a KAPA SYBR Fast qPCR Kit (KAPA Biosystems) and its size distribution was analyzed by a MultiNA. WTA, TCRseq, and Sample tag libraries were sequenced on an Illumina Novaseq 6000 S4 flowcell (67 bp read 1 and 140 bp read 2) using NovaSeq 6000 S4 Reagent Kit v1.5 to a depth of approximately 20,000 reads, 7,000 reads, and 10,000 reads per cell, respectively.