title: GSM4673032: P1_Tumor_0003; Homo sapiens; OTHER
library_construction_protocol: 10 ml of patient's blood was taken at four time point (P1; day0, P2; day21-27, P3; day39-55, P4; disease progression). PBMCs were prepared by density gradient centrifugation by Ficoll®-Paque Plus (GE Healthcare Japan). The PBMCs were separated in two samples, then CD8+ T cell and CD4+ T cell were enriched by using CD8 Microbeads or CD4 Microbeads for human (Milteny Biotec Inc., Bergisch Gladbach, Germany). The purity was checked by FACSCanto II (BD Biosciences) with staining with CD8a-APC (clone RPA-T8, TONMO Biosciences, San Diego, CA, USA ), CD3-FITC (clone UCHT1, TONBO Biosciences, San Diego, CA, USA), CD4-PE (clone SK3, BioLegend), and Ghost Dye Red780 (TONBO Biosciences). The purity of enriched cells was routinely more than 95%. Tumor biopsy was conducted on before (day0) and after the treatment (day39-day55). Presence or absence of tumor tissue in biopsies was determined pathologically. Tumor biopsies were homogenized in TRIzol (Ambion, Carlsbad, CA, USA). RNA was extracted from each sample using the RNeasy mini kit (QIAGEN, Hilden, Germany, # 74106) and amounts and purity measured with the Agilent 2200 TapeStation (Agilent Technologies, Santa Clara, CA, USA). Five-micrograms of total RNA was diluted with 1 mL of cell lysis buffer and used for TCR sequencing. TCR sequencing libraries for next-generation sequencing were prepared according to the previous report (GSE115425) with some modifications. PolyA RNAs were isolated according to a previous report with some modifications (GSE110711). 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 first PCR mixture comprised 0.8 μL of 10 μM primer mix (trP1, Trac_ex and Trbc_ex), 4.2 μL of dH2O, and 5 μL of KAPA Hifi Hotstart ReadyMix (KAPA Biosystems, Wilmington, MA, USA, #KK2602). The thermal cycling conditions were programmed as follows: 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. Next, 10 μL of the first-PCR products were 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 15 μL of DW. The second PCR mixture consisted of 1.75 μL of 10 μM primer mix (5' WTA, Trac_in-Bio, and Trbc_in-Bio), 10.75 μL of the template and 12.5 μL of KAPA Hifi Hotstart ReadyMix. The thermal cycling conditions were the same as the first PCR except for the cycle number; 21 cycles. 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 18 μL of DW. Fragmentasion and adaptor ligation were performed using 10-20ng of second-PCR product as a template with NEBNext FS DNA Library Prep Kit (New England Biolabs, #E7805) with some modifications; NEBNext Adaptor for Illumina in “Adaptor Ligation” step was substituted by 5uM Adaptor P1, and the reaction volume was 1/4 of the recommended in all steps. Adaptor-ligated DNA was purified using Agencort AM Pure XP kit (Bexkman-Coulter) at a 0.8:1 ratio of beads to sample, and eluted in 20 μL of DW. The third PCR was carried out using barcoded primers to enrich the TCR cDNA flanked with sequencing adapters. The third PCR mixture consisted of 1 μL of 10 μM trP1 primer, 1 μL of 10 μM IonA-BC-Trbc primer, 3 μL of template and 5 μL of NEBNext Ultra II Q5 Master Mix (accessory of NEBNext FS DNA Library Prep Kit). The thermal cycling conditions were programmed as follows: denaturation at 98°C for 30 sec, 10 cycles of denaturation for 10 sec at 98°C, annealing and extension for 1min 15 sec at 65°C, followed by a 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 1.0:1 ratio of beads to sample, and eluted with 20 μL of Tris-HCl (pH 8.0). Amplified TCR libraries were quantified using a KAPA SYBR Fast qPCR Kit (KAPA Biosystems, #KK4621) 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 540 Kit Chef, an Ion 540 Chip kit, and an Ion Genestudio S5 Sequencer (Thermo Fisher Scientific, # A27759, #A27766, # A38194) according to the manufacturer's instructions, except the input library concentration (65 pM) and flow number (500).
attributes.tag: GEO Accession
attributes.value: GSM4673032