Sbs kits

PBMCs from subjects with T1D, RA, RCC and age/gender-matched HC were stimulated with antibodies against CD3 (1 µg/ml plate-bound, UCHT1) and CD28 (2 µg/ml plate-bound, CD28.2) for 16 hours with and sorted for memory (CD45RO⁺) CD8 T cells that either co-expressed KLRG1 and TIGIT or lacked both markers as a comparison population using the cell sorting panel (Supplementary Table 1). Sytox Green (1:1000, Invitrogen) was added to samples prior to acquisition to differentiate dead cells. For comparisons across cells of differing antigen specificities, cells from HC were enriched for CD8 T cells following the 16-hour stimulation protocol, then stained with Class I pentamer to identify CMV, Epstein-Bar Virus (EBV), and Flu antigens (Supplementary Table 1) as described below.
The indicated populations were sorted directly into SMARTer v3 or SMARTseq v4 lysis reagents (Clontech). Cells were lysed and cDNA was synthesized. After amplification, sequencing libraries were prepared using the Nextera XT DNA Library Preparation Kit (Illumina) according to C1 protocols (Fluidigm). Barcoded libraries were pooled and quantified using a Qubit^® Fluorometer (Life Technologies). Single-read sequencing of the pooled libraries was carried out on a HiSeq2500 sequencer (Illumina) for 74 cycles, using TruSeq v3 or v4, and SBS kits (Illumina). Target read depths were ~5-10 million raw reads per sample.

WGBS libraries were prepared following Illumina’s “Whole-Genome Bisulfite Sequencing for Methylation Analysis” protocol. Briefly, 1 μg of genomic DNA was spiked with 0.5% unmethylated lambda DNA and sonicated to generate fragments of size between 150 to 300 bp. Library preparation was performed using Illumina’s Paired-end DNA Sample Prep Kit (discontinued, Illumina, San Diego, CA, USA) according to the manufacturer’s protocol. The size-selected libraries were then subjected to bisulfite conversion as previously described [55 (link)]. Adaptor-ligated bisulfite-treated DNA was enriched by 10 cycles of PCR amplification using the PfuTurbo Cx Hotstart DNA Polymerase (Stratagene, La Jolla, CA, USA). Qualitative and quantitative checks of the libraries were performed using Agilent’s High sensitivity DNA kit (Agilent, Santa Clara, CA, USA) and KAPA Library quantification kit (KAPA Biosystems, Wilmington, MA, USA). Three lanes of paired-end 100 bp sequencing was performed for each of the libraries on the Illumina HiSeq2500 platform using the TruSeq v3 cluster kits and SBS kits (Illumina) to achieve coverage ranging between 25 × and 30 ×.