GSM5289600: day13_dLN_CD8_right_5_1; Mus musculus; OTHER
Tumor was divided into two and treated individually. Each tumor was cut into small fragments and digested for 45 minutes at 37°C with 0.1% collagenase (FUJIFILM Wako, Osaka, Japan). The cells were then subjected to 40% Percoll PLUS (Cytiva, Marlborough, MA) and leukocytes were recovered from the cell pellet. ACK buffer was added to the cells for lysing of red blood cells. The extracted dLN was cut into small fragments and mashed using a cell strainer. The cell number was determined using Flow-Count fluorospheres (Beckman Coulter, San Diego, CA) and a CytoFLEX S flow cytometer (Beckman Coulter). Cells were then stained with a mix of Fc Block (anti-mouse CD16/CD32 mAb; clone 2.4G2, BioXcell) and fluorophore-conjugated anti-mouse mAbs as indicated in Table S1. After T cells were enriched magnetically, CD8+ T cells, CD4+ T cells, CD8+ CD44hi T cells or CD4+ CD44hi T cells were sorted using FACS Aria II or Aria III (BD Biosciences, San Jose, CA). Nonviable cells and IVS-CD45.2 positive cells were excluded from the sorting gate based on forward and side scatter profiles and propidium iodide staining. Purity of sorted cells was always over 95%. 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, 5 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 under the same condition as first PCR. 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.