title: Illumina HiSeq 2500 paired end sequencing; Mapping T cell responses to native and neo- islet antigen epitopes in at risk and type 1 diabetes subjects
design: Mapping T cell responses to native and neo- islet antigen epitopes in at risk and type 1 diabetes subjects
library_construction_protocol: Each experiment was conducted using blood obtained from 2 individuals with type 1 diabetes; PBMCs at 2x106/ml were added to 20 wells of a 48-well plate, pool 1 peptides were added at a final concentration of 10ug/ml; this was repeated for an additional 20 wells where pool 2 was added at a final concentration of 10ug/ml. Pediacel, was used at positive control and 1μL/mL was added to a well containing cells at 2x106/ml and media alone was used as a negative control. Anti-CD40 (Biolegend, clone G28.5, 303611) was added at a final concentration of 2 ug/ml to each well and the cells were incubated for 18-20h. The cells were then harvested and washed with phosphate buffered saline (PBS). An antibody staining master mix consisting of anti-CD19, (M5E2, 561391), anti-CD3 (SK7, 641415), anti-CD4 (SK3, 345768), anti-CD45-RO (UCHL1, 337168), anti-CD95 (DX2, 561978), anti-CD154 (TRAP1, 555700), anti-CD69 (FN50, 555530) (BD Biosciences, Oxford, UK) and anti-CD27 ((O323, 302830) Biolegend, London UK) was prepared and spilt into 5 tubes. Anti-human Hashtag 1 (TotalSeqC 0251), anti-human Hashtag 2 (TotalSeqC 0252), anti-human Hashtag 3 (TotalSeqC 0253), anti-human Hashtag 4 (TotalSeqC 0254) and anti-human Hashtag 5 (TotalSeqC 0255), (Biolegend, London, UK) were added at a concentration of 0.1μg/μl to each of the 5 tubes. The hashtags were used to barcode the samples as follows: patient, 1 pool 1 (Hashtag 1); patient 1 pool 2 (Hashtag 2) patient 2 pool 1 (Hashtag 3); patient 2 pool 2 (Hashtag 4) and Pediacel (patient 1 and 2; Hashtag 5). The hashtag barcoded cells were incubated for 20 mins in the dark and washed 3 times in FACS buffer (PBS with 3% FCS and 2% human (both from AB Sigma, UK). CD4+ cells responding to stimulation were identified by co-expression of CD69 and CD145 were sorted into 35μL PBS using a BD FACSAria III cytometer  Sorted cells were characterised using the 10x Chromium Controller Single-cell Immune Profiling system which simultaneously defines gene expression and T cell receptor profiles from single cells in the same sample; this was conducted at the Genomics facility at Guy's and St Thomas' Biomedical Research Centre. Briefly, single-cell suspensions were used to generate full-length cDNA in gel beads (containing Unique Molecular identifiers - UMI) in an emulsion master mix from 5' V1 chemistry. The cDNA was amplified and subsequently size selected at the clean-up stage: smaller fragments were separated and stored for feature-barcoding/cell-hashing library preparation and the large fragments for gene expression (GEX) and T cell VDJ according to manufacturer's instructions. The GEX libraries were enzymatically fragmented and indexed while the T cell library was first enriched (using Chromium Single Cell V(D)J Enrichment Kit, Human T Cell, PN-1000005) and then fragmented and indexed for further sequencing.