Single cell RNA-sequencing of T cells to examine barcode swapping

T cells from OT-I mice were stimulated with 4 different peptide ligands for 6 hours and sorted into two 96-well plates. Cells on each plate were barcoded using a mutually exclusive 8-by-12 set of indexes, such that indexes present on plate 1 were completely absent from plate 2 and vice versa. Libraries from each plate were pooled in equimolar quantities and sequenced on an Illumina HiSeq 4000. Libraries were demultiplexed allowing for all pairs of indexes, including the expected combinations (pairs of barcodes used within each plate) and unexpected combinations (pairs containing one barcode from each plate). This was repeated after sequencing the same pool of libraries on the HiSeq 2500.