Chromium chip b

scRNA-seq experiments were performed by the Brigham and Women's Hospital Single Cell Genomics Core. Sorted viable human T cells were stained with a distinct barcoded antibody (Cell-Hashing antibody, TotalSeq-A; BioLegend), as previously described.⁵⁸ (link) Next, 7,500 cells from each condition were resuspended in 0.4% BSA in PBS at a concentration of 1,000 cells per μL, pooled together, and then loaded onto a single lane (Chromium chip B, 10X Genomics) followed by encapsulation in a lipid droplet (Single Cell 3′ kit V3, 10X Genomics) followed by cDNA and library generation according to the manufacturer's protocol. mRNA library was sequenced to an average of 50,000 reads per cell and hashtag-oligos (HTOs) (Cell Hashing antibodies) library sequenced to an average of 5,000 reads per cell, both using Illumina Novaseq. scRNA-seq reads of 14,446 cells were processed with Cell Ranger v3.1,⁵⁹ (link) where read quantification was performed using the STAR aligner⁶⁰ (link) against the GRCh38 transcriptome. There were 33,908 mean reads per cell, 21,577 genes were detected, and there were 4,855 median unique molecular identifier (UMI) counts per cell. Single-cell data analysis was performed using R version 4.0.3, and code is available from https://github.com/aedin.

Dissociated, sorted cells were stained with Trypan blue and the Countess II Automated Cell Counter (Thermo Fisher Scientific) was used to assess both cell number and viability (74–82%). Following QC, the single-cell suspension was loaded onto Chromium Chip B (10x Genomics PN 2000060) and Gel Beads in emulasion generation, cDNA synthesis, cDNA amplification, and library preparation of 4,500–9,300 cells proceeded using the Chromium Single Cell 3′ Reagent Kit v3 (10x Genomics PN 1000075) according to the manufacturer’s protocol. cDNA amplification included 11 cycles, and 44–87 ng of the material was used to prepare sequencing libraries with 12 cycles of PCR. Indexed libraries were pooled equimolar and sequenced on a NovaSeq 6000 in a PE28/91 paired end run using the NovaSeq 6000 SP, S1, or S2 Reagent Kit (100 cycles; Illumina). An average of 201 million paired reads was generated per sample.