Totalseq a

For isolation of CD8 T cells 10 days after infection, single-cell suspensions were generated from four mice per recipient group by macerating spleens through nylon filters. CD8 T cells were enriched from these suspensions using a Stemcell EasySep™ Mouse CD8 T Cell Isolation Kit (#19853). These samples were stained to block Fc receptors then stained with antibodies and live/dead stain (LIVE/DEAD™ Fixable Violet Dead Cell Stain Kit, ThermoFisher # L34955) for 30 minutes on ice shielded from light. The antibodies used for cell surface staining from BioLegend were as follows; PE anti-mouse CD8β Antibody (YTS156.7.7), APC anti-mouse CD45.1 Antibody (A20) and APC anti-rat CD90/mouse CD90.1 (Thy-1.1) Antibody (OX-7). Samples were subsequently washed twice and ~1X10⁶ congenically marked OT-I cells were purified using fluorescence activated cell sorting for each group of recipients. The samples purified in this way from each group of recipients were then suspended in 100μL buffer and labeled with 1μg per sample of the following Total-seq A antibodies from BioLegend: TotalSeq™-A0198 anti-mouse CD127 (A7R34), TotalSeq™-A0250 anti-mouse/human KLRG1 (2F1/KLRG1), TotalSeq™-A0073 anti-mouse/human CD44 (IM7) and TotalSeq™-A0112 anti-mouse CD62L (MEL-14). Samples were labeled for 30 minutes on ice and subsequently washed with 1mL PBS twice.

For single cell RNA-seq, pro-T cells obtained from OP9-Dll1 culture were stained with surface antibodies followed by hashtag oligo labeling with TotalSeq A (BioLegend) anti-Mouse Hashtag 1-8 (1:50, in separate samples). After FACS sorting the target cells, samples were washed with 1X HBSS supplemented with 10% FBS and 10 mM HEPES pH 7.3-7.5 and resuspended to 1x10⁶ cells/1mL concentration. Then, 16,000 cells were loaded into a 10X Chromium v3 lane, and the subsequent preparation was conducted following the instruction manual of 10X Chromium v3.

C&R libraries were prepared using NEBNext ChIP-Seq Library Preparation Kit (NEB) by following a previously published protocol⁶¹ . ChIP-seq libraries were prepared using a NEBNext ChIP-Seq Library Preparation Kit (NEB) according to the manufacturer’s protocol. For generating ATAC-seq libraries, tagmented DNA was PCR amplified (7-8 cycles) by determining the number of amplification cycle for each sample using qPCR^{58 ,59} and size-selected for 150 bp – 3000 bp range using AMPure XP beads (Beckman Coulter A63880). Single cell RNA-seq cDNA libraries were prepared using 10X Chromium 3’ capture v3 kit. The single-cell hashtag oligo library was prepared by following the BioLegend TotalseqA guide. After the library preparation, the sequencing was performed with paired-end sequencing of 50 bp (C&R, ChIP-seq, and ATAC-seq) or 150 bp (single-cell RNA-seq) using HiSeq4000 by Fulgent Genetics, Inc. (Temple City, CA) or NextSeq by the California Institute of Technology Genomics core. Libraries were sequenced to the following targeted read depths: C&R libraries, 10 million reads; ChIP-seq libraries, 30 million reads; ATAC-seq libraries, 40 million reads. Single cell RNA-seq cDNA libraries were sequenced to a targeted depth of 65,000-70,000 reads per cell and hashtag oligo libraries were sequenced for 2,000-2,500 reads per cell.