GSM3178047: control_TCRb_dLN44hi_5; Mus musculus; OTHER
dLN, peripheral blood and tumor were collected at day 14 post tumor inoculation. For peripheral blood, red blood cells were lysed using Ammonium-Chloride-Potassium lysis buffer. Tumor were cut into small fragments and digested for 45 min at 37 °C with 0.2% collagenase (Wako, Osaka, Japan) and 20 kU/mL DNase I (Sigma-Aldrich, St Louis, MO), and the recovered from tumor were then subjected to 40/70 % Percoll (GE Healthcare, Buckinghamshire, UK) gradient and leukocytes were recovered from the interphase. Resulting cells were pretreated with Fc Block (anti-mouse CD16/CD32 mAb; clone 2.4G2, BioXcell, West Lebanon, NH, USA), then stained with a mixture of fluorophore-conjugated anti-mouse mAbs. CD8+ T cells or CD8+ CD44hi T cells were purified by cell sorting. Then, we performed TCR sequencing analysis on these cells. TCR sequencing libraries for next generation sequencing were prepared from the mRNA of sorted CD8+ T cell samples. PolyA RNAs were isolated and amplified from sorted T cells according to a previous report (Shichino, et al, GSE110711). To amplify the TCR cDNA containing complementarity determining region 3 (CDR3), nested PCR of the TCR locus was performed as follows. The first PCR mixture comprised 2.8 μL of distilled water (DW), 0.2 μL of 10 μM primer mix (5′ WTA, Trac_ex and Trbc_ex), 2 μL of cDNA template, and 5 μL of 2x Q5 High-Fidelity DNA Polymerase (New England Biolabs, MA, USA, NEB #M0491). The thermal cycling conditions were programmed as follows: denaturation at 98°C for 30 s, 10 cycles of denaturation for 10 s at 98°C, annealing and extension for 75 s at 65°C, followed by final extension at 65°C for 5 min. Next, 10 μL of the first-PCR products was used for purification with an Agencort AM Pure XP kit (Bexkman-Coulter, CA, USA, #A63881) at a 0.7:1 ratio of beads to sample, and eluted with 10 μL of DW. The second PCR mixture consisted of 7.25 μL of DW, 0.2 μL of 10 μM primer mix (5′ WTA, Trac_in-Bio and Trbc_in-Bio), 4 μL of purified PCR product and 12.5 μL of 2x Q5 High-Fidelity DNA Polymerase (New England Biolabs). The thermal cycling conditions were programmed as follows: denaturation at 98°C for 30 s, 18-23 cycles of denaturation for 10 s at 98°C, annealing and extension for 75 s at 65°C, followed by a final extension at 65°C for 5 min. Next, 25 μL of the second-PCR products were purified using Agencort AM Pure XP kit (Bexkman-Coulter) at a 0.7:1 ratio of beads to sample, and eluted in 16 μL of DW. The purified PCR products were sheared randomly using NEBNext dsDNA fragmentase (New England Biolabs, #M0348). The fragmentation reaction mix consisted of 2.3 μL of DW, 0.2 μL of 10 mg/ml BSA, 1 μL of 100 mM MgCl2, 2 μL of 10x reaction buffer, 10 μL of 8 ng/μL of PCR product, and 2 μL of Fragmentase. The fragmentation reaction was incubated at 37°C for 30 min, and then 5 μL of 0.5 M EDTA was added to stop the reaction. The sheared PCR product was then purified and subjected to size selection using the Agencort AM Pure XP kit (Bexkman-Coulter) at a 0.8:1 ratio of beads to sample to remove large fragments, a 0.8:1 ratio of beads to sample to remove the smaller fragments, and eluted with 20 μL of Tris-HCl (pH 8.0). To capture the TCR cDNA containing the end of Constant region, the purified PCR products were incubated with 30 μL of Dynabeads M-270 Streptavidin (Thermo Fisher Scientific, MA, USA, #DB65305) for 30 min at room temperature (r.t.), washed 3 times 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), and DW. The captured TCR cDNA was repaired using NEBNext Ultra II End Repair/ dA-Tailing Module (New England Biolabs, # E7546L). The repair reaction mix contained 1.2 μL of reaction buffer, 8.3 μL of beads re-suspended with DW, and 0.5 μL of enzyme mix. The repair reaction was incubated at 20°C for 30 min, then washed once with lysis buffer [1% LiDS, 100 mM Tris-HCl (pH 7.5), 500 mM LiCl, 10 mM EDTA, and 5 mM DTT], 3 times with B&W-T buffer, and once with Tris-HCl (pH 8.0). The repaired TCR cDNA was attached to the sequencing adaptor using the DNA Ligation Kit ＜Mighty Mix＞ (TaKaRa, Shiga, Japan, #6023). The ligation reaction mix consisted of 1 μL of 10 μM P1 adaptor, 6.5 μL of beads re-suspended in Tris-HCl (pH 8.0), and 15 μL of enzyme mix. The ligation reaction was incubated at 16°C for 60 min using a thermal cycler with the cover open, washed once with lysis buffer, 3 times with B&W-T buffer, and once with Tris-HCl (pH 8.0). The third PCR were carried out using barcoded primers to enrich the TCR cDNA flanked with sequencing adapters. The third PCR mixture consisted of 6 μL of DW, 1.25 μL of 10 μM trP1 primer, 1.25 μL of 10 μM IonA-BC-Trac or IonA-BC-Trbc primer, 4 μL of beads resuspended in DW and 12.5 μL of 2x Q5 High-Fidelity DNA Polymerase (New England Biolabs). The thermal cycling conditions were programmed as follows: denaturation at 98°C for 30 s, 9 cycles of denaturation for 10 s at 98°C, annealing and extension for 75 s at 65°C, followed by final extension at 65°C for 5 min. The PCR product was purified and subjected to size selection using Agencort AM Pure XP kit (Bexkman-Coulter,) at a 0.75:1 ratio of beads to sample to remove large fragments, a 0.65:1 ratio of beads to sample for TCRα or 0.5:1 ratio for TCRβ to remove smaller fragments, and eluted with 20 μL of Tris-HCl (pH 8.0). Amplified TCR libraries were quantified using a KAPA Library Quantification Kit (NIPPON Genetics, Tokyo, Japan, KK4827) and size distribution was analyzed by agarose electrophoresis and SYBR Gold staining (Thermo Fisher Scientific, #S11494).   Final TCR libraries, whose lengths were 200–300 base pairs, were pooled and sequenced using an Ion Hi-Q Chef kit, an Ion PI v3 Chip kit, and an Ion Proton Sequencer (Thermo Fisher Scientific, # A27198, #A26771, # 4476610) according to the manufacturer's instructions, except the input library concentration (100 pM) and flow number (500).