Totalseq b0301 b0306

Single-cell suspensions of 11 Myc;Ptenfl tumors from 6 mice were obtained by enzymatic digestion. Tissue was manually minced using scissors, followed by a 30–60 min enzymatic digestion with 2.0 mg/ml collagenase A (Roche), 1.0 mg/ml Hyaluronidase (Worthington), and 50 U/ml DNase I (Roche) in serum-free Dulbecco’s modified eagles medium (DMEM) (Invitrogen) and Rock inhibitor at 37 °C using continuous stirring conditions. Single-cell suspensions from tumor digests were prepared by passing tissue through 40-mm nylon strainers (BD Biosciences). Single-cell suspensions from individual tumors were then labeled with hashtag oligonucleotides following the manufacturer’s protocol (TotalSeq B0301–B0306, Biolegend). Each individual tumor sample was counted and then pooled at an equal cell ratio before being split into two replicates for library preparation with the Chromium Single Cell 3’ V3 (10× Genomics) following the manufacturer’s protocol with a targeted recovery of 20,000 cells per library. Libraries were sequenced on an Illumina NovaSeq. BCL files were converted to fastq format with bcl2fastq2 (Illumina) and then aligned to mouse genome build mm10-2020-A (10× Genomics) using Cellranger (10× Genomics, version 6.0.2).

The BioLegend TotalSeq B0301-B0306 was used, containing CD45 (clone 30-F11) and MHC-I (clone M1/42). The M1/42 clone (anti-MHC I) is reported to recognize cells from C57BL/6 (B6) mice, which have the H-2b haplotype, but it was not tested for cells from FVB mice, which have the H-2q haplotype. To test whether M1/42 binds FVB poorly or not at all, we stained both strains with fluorophore-conjugated CD45 (clone 30-F11) and MHC-I (clone M1/42) (Supplemental Fig. S11C). Single-cell suspensions were stained in FACS buffer (1× PBS, with 2% FBS and 0.1% NaN3) at 4 °C, for 20 min, in the dark. Antibodies used are as follows: CD45 (clone 30-F11) and MHC-I (clone M1/42), EpCAM (clone G8.8) and TER-119 (clone TER-119). Cells were run on a BD FACS Symphony A5 (BD Biosciences). Data were analyzed using FlowJo 10.6 (Tree Star, Inc., RRID:SCR_008520). The M1/42 clone stained all B6 cells. CD45+ FVB stained less brightly than B6 cells but were clearly MHC I+. Some FVB cells were not stained, but of the MHC I negative cells, nearly all were TER-119+, indicating they are red blood cells that are expected to lack MHC I staining. Thus, M1/42 appears to cross-react to FVB cells, with reduced staining intensity but was sufficiently reactive to use TotalSeq (Biolegend) reagents on FVB cells (Supplemental Fig. S11C).