GSM8322435: EnhDel CD8+ T cells, Day 9, Slamf6+TIM3-, sample 4; Mus musculus; RNA-Seq
After CD8+ T cells were isolated from spleen, cells were sorted: (1) live CD8b+ CD44+ CD45.1+ CD45.2- Slamf6+ TIM3-, (2) live CD8b+ CD44+ CD45.1+ CD45.2- Slamf6- TIM3+ CX3CR1-, (3) live CD8b+ CD44+ CD45.1+ CD45.2+ Slamf6+ TIM3-, (4) live CD8b+ CD44+ CD45.1+ CD45.2+ Slamf6- TIM3+ CX3CR1-. Samples from eight recipient mice were then pooled in pairs to create four biological replicate samples. Cells were stored at -80C, then cell lysates were thawed on ice. RNA was purified using 2.2x volume of RNAClean XP beads (Beckman Coulter, A63987). Samples were incubated for 10 minutes at room temperature and then placed on a DynaMag-96 side skirted magnet (Invitrogen, 12027) for 5 minutes, followed by supernatant aspiration and two washes with 80% ethanol. Dried beads were resuspended in 4μL of Mix-1 containing 1μL (10 μM) primer (IDT, DNA oligo) 5′–AAGCAGTGGTATCAACGCAGAGTACT30VN-3′, 1μL (10 mM) dNTPs (Thermo-Fisher, R0192), 1μL (4U/μL) Recombinant RNase Inhibitor (Takara Bio, 2313A), and 1μL nuclease-free water per sample, and plates were placed in a thermocycler for 3 minutes at 72°C for RNA denaturation. Samples were immediately placed on ice. Reverse transcription was performed by adding 7μL of Mix-2 containing 0.75μL nuclease-free water, 2μL 5X Maxima RT buffer (Thermo Scientific, EP0752), 2μL (5M) betaine (B0300-1VL), 0.9μL (100mM) MgCl2 (Thermo Scientific, AM9530G), 1μL (10μM) TSO primer (IDT, RNA oligo) 5′-AAGCAGTGGTATCAACGCAGAGTACATrGrG+G-3′, 0.25μL (40U/μL) Recombinant RNase Inhibitor (Takara Bio, 2313A), and 0.1μL (200U/μL) Maxima H Minus Reverse Transcriptase (Thermo Scientific, EP0752); plates were incubated in a thermocycler for 90 minutes at 50°C followed by 5 minutes at 85°C. PCR preamplification was performed next by adding 14μL of Mix-3 containing 1μL nuclease-free water, 0.5μL ISPCR primer (IDT, DNA oligo) 5′-AAGCAGTGGTATCAACGCAGAGT-3′, and 12.5μL 2X KAPA HiFi HotStart ReadyMix (Roche, 07958935001); whole-transcriptome amplification was done by incubating in a thermocycler for 3 minutes at 98°C followed by 17 to 20 cycles of [98°C for 15 seconds, 67°C for 20 seconds, 72°C for 6 minutes] depending on cell number input, followed by final extension at 72°C for 5 minutes. Reaction cleanup and primer dimer removal was performed by incubating samples with 20μL Agencourt AMPure XP beads (Beckman Coulter, A63881) for 5 minutes, placing the plate on a DynaMag-96 side skirted magnet for 5 minutes, followed by supernatant removal and two washes with 70% ethanol and resuspension of dried beads in 20μL of TE buffer (Tekanova, T0228). This step was repeated for complete primer dimer removal. cDNA concentration was determined using the Qubit dsDNA high sensitivity assay kit (Thermo Scientific, Q32854), and cDNA size distribution was evaluated via TapeStation using a High Sensitivity D5000 Screentape (Agilent, 5067-5592). Samples were diluted to a concentration of 0.1-0.2ng/μL. The cDNA library was generated using the Nextera XT Library prep kit (Illumina, FC-131-1096) according to the manufacturer's protocol using barcodes from the Nextera XT Index Kit v2 Set A (Illumina, FC-131-2001). Barcoded samples were pooled and cleaned using Agencourt AMPure XP beads (Beckman Coulter, A63881); concentration was determined using the Qubit dsDNA high sensitivity assay kit and size distribution was determined using the TapeStation High Sensitivity D5000 Screentape. The pooled library containing 36 samples was sequenced on a NextSeq 1000 sequencer (Illumina) using the P2 (100 cycle) v3 reagent kit (Illumina, 20046811) using paired-end 50-base reads.