GSM5800311: CS1_S425; Homo sapiens; RNA-Seq
Blood KIR+ CD8+ T cells (Live CD3+CD56–CD8+TCRαβ+KIR+ cells) were sorted into 96-well plates and cDNA synthesis was performed using the Smart-seq2 protocol Illumina sequencing libraries were prepared using a Mosquito HTS liquid handler (TTP Labtech). In brief, tagmentation was carried out on 0.4 μl double-stranded cDNA using the Nextera XT DNA Library Preparation Kit (Illumina, FC-131-1096). Each well was mixed with 0.8 μl Nextera tagmentation DNA buffer (Illumina) and 0.4 μl Amplicon Tagment Mix (Illumina), then incubated at 55°C for 10 min. The reaction was stopped by adding 0.4 μl Neutralize Tagment Buffer (Illumina) and centrifuging at room temperature at 3000 g for 5 min. Indexing PCR reactions were performed by adding 0.8 μl of pre-mixed 5 μM i5 and i7 unique dual indexing primers (IDT, customized) and 1.2 μL of Nextera NPM mix (Illumina). PCR amplification was carried out on a C1000 Touch™ Thermal Cycler with 384-Well Reaction Module (Bio-rad) using the following program: 1) 72°C for 3 min, 2) 95°C for 30 s, 3) 12 cycles of 95°C for 10 s, 55°C for 30 s and 72°C for 1 min, and 4) 72°C for 5 min.