title: GSM4673099: RNA_P4_Tumor_1_0002; Homo sapiens; RNA-Seq
library_construction_protocol: 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 RNA sequencing. PolyA RNAs were isolated according to a previous report with some modifications (GSE110711). Beads with containing cDNA was resuspended with whole transcript amplification mixture comprised 2 μL of 10 μM primer mix (trP1 and 3'WTA), 10.5 μL of dH2O, and 12.5 μL of KAPA Hifi Hotstart ReadyMix (KAPA Biosystems). The thermal cycling conditions were programmed as follows: denaturation at 95°C for 3 min, 12 cycles of denaturation for 20 sec at 98°C, annealing for 15 sec at 65°C and extension for 5 min at 72°C, followed by a final extension at 72°C for 5 min. Next, 25 μL of the first-PCR products were used for purification with an Agencort AM Pure XP kit (Bexkman-Coulter) at a 0.7:1 ratio of beads to sample, and eluted with 15 μL of DW. Fragmentasion and adaptor ligation were performed using 10-60ng of second-PCR product as a template with NEBNext FS DNA Library Prep Kit (New England Biolabs) with some modifications; NEBNext Adaptor for Illumina in “Adaptor Ligation” step was substituted by 1.5uM CS1 adaptor, and the reaction volume was 1/4 of the recommended in all steps. Adaptor-ligated DNA was added with 7.125 uL of 10x TE, then purified using Agencort AM Pure XP kit (Bexkman-Coulter) at a 0.4:1 ratio of beads to sample to remove large fragments, a 0.7:1 ratio of beads to sample to remove smaller fragments, and eluted with 20 μL of Tris-HCl (pH 8.0). The second PCR was carried out using barcoded primers to enrich the TCR cDNA flanked with sequencing adapters. The second PCR mixture consisted of 2.5 μL of 10 μM trP1 primer, 2.5 μL of 10 μM IonA-BC primer, 7.5 μL of template and 12.5 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, 11 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 0.7:1 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 RNAseq 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 (550).
attributes.tag: GEO Accession
attributes.value: GSM4673099