Truseq v3 cluster and sbs kits

After flow sorting, single cells were captured on the Fluidigm C1 Single-Cell Auto Prep System (C1), lysed on chip, and subjected to reverse transcription and cDNA amplification using the SMARTer Ultra Low Input RNA Kit for C1 System (Clontech, Mountain View, CA). Sequencing libraries were prepared using the Nextera XT DNA Library Preparation Kit (Illumina, San Diego, CA) according to C1 protocols (Fluidigm). Barcoded libraries were pooled and quantified using a Qubit Fluorometer (Thermo Scientific Life Technologies, Grand Island, NY). Single-read sequencing of the pooled libraries was carried out either on a HiScanSQ or a HiSeq2500 sequencer (Illumina) with 100-base reads, using TruSeq v3 Cluster and SBS kits (Illumina) with a target depth of >2.5 M reads. Sequences were aligned to the UCSC Human Genome Assembly version 38, gene expression levels quantified using RSEM and TPM values loaded into R for analyses. We used the MAST analysis platform(23 (link)) for subsequent singe-cell analysis steps. Quality control parameters for the single-cell RNAseq data included expression of at least 500 genes, alignment rate bigger than 80%, library size > 10000 and exon rate bigger than 30% (all 4 criteria have to be met to for a single cell to be included). MAIT cells were analyzed directly after sorting and were not stimulated prior to single-cell RNAseq processing.