Hiseq 400

Single-cell capture and library construction was performed with the 10x Genomics Chromium Single Cell 5’ kits v.1.0 (product codes 1000014, 1000020, and 1000151) with TCR enrichment (1000005) for cohort MDA1 and 3’ V.2 kits (product codes120237, 120236, 120262) for MDA2, according to the manufacturer’s instructions. Briefly, cells were loaded into the Chromium Single Cell Chip A for a recovery target of 10,000 cells. Reverse transcription was performed on a BioRad T100 thermal cycler, and the barcoded cDNA was purified with Dynabeads (Thermo Fisher Scientific, 37002D) prior to 14 cycles of cDNA amplification. Of this transcriptome cDNA, 2 μL was used for TCR enrichment and subsequent TCR library construction in the MDA1 cohort. Per the manufacturer’s protocol, up to 50 ng (or 20 μL) of transcriptome cDNA was used for single-cell library construction. Transcriptome libraries were sequenced on a HiSeq 400 (Read 1, 26 cycles; Index 1, 8 cycles; Read 2, 91 cycles), and TCR libraries were pooled and sequenced 150 cycles paired-end on a MiSeq (Illumina). For transcriptome libraries, median sequencing depth for each sample was targeted for a median of 30,000 reads/cell; for TCR libraries, 1,000 reads/cell. Read counts can be found in Supplementary Table S3.

Immediately post-sorting, Ghost dye^- CD45⁺ CD3⁺ CD4⁺ GFP⁺ skin cells were run on the 10X Chromium (10X Genomics (27 (link))) platform and library preparation was performed by the Institute for Human Genetics at UCSF following the recommended protocol for the Chromium Single Cell 3’ Reagent Kit (v2 Chemistry). Libraries were run on the HiSeq400 for Illumina sequencing. Post-processing and quality control were performed by the Genomics Core Facility in the Institute for Human Genomics at UCSF using the 10X Cell Ranger package (v1.2.0, 10X Genomics). Reads were aligned to mm10 reference assembly (v1.2.0, 10X Genomics). Primary assessment using this software showed 2,058 median unique molecular identifiers (UMIs, transcripts) per cell and 1,035 median genes per cell sequenced to 91.6% sequencing saturation with 88,229 mean reads per cell.

For RNAseq, tissues were homogenized TRIzol (1 mL per 100 mg of tissue) and incubated for 24 h. Samples were then incubated in chloroform for 5 min and centrifuged at 12,000 g for 15 min at 4 °C. The aqueous upper phase containing RNA was transferred to a tube containing an equal volume of 70% ethanol and vortexed. RNA was treated with DNases and isolated using the PureLink RNA isolation kit (ThermoFisher Scientific). Total RNA was assessed for quality using an Agilent Tapestation 4200, and 1 μg of RNA from samples with an RNA Integrity Number (RIN) greater than 7.0 were used to generate RNA sequencing libraries using the TruSeq Stranded mRNA Sample Prep Kit (Illumina, San Diego, CA). Samples were processed following the manufacturer’s instructions, modifying RNA shear time to five min. Resulting libraries were multiplexed and sequenced with 75 basepair (bp) single reads (SR75) to a depth of approximately 25 million reads per sample on an Illumina HiSeq400. Samples were demultiplexed using bcl2fastq Conversion Software (Illumina, San Diego, CA). QC and RNAseq were conducted at the IGM Genomics Center, University of California, San Diego, La Jolla, CA.