Ter119 pe cy7

CD23 (B3B4)-PE and -BV421[1:200], TCRβ (H57-597)-PE[1:400], Ter-119-PE[1:400], CD138 (281-2)-PE[1:400] and XBP1s (Q3-695)-PE-CF594[1:200] were purchased from BD Biosciences. IgD (11-26.2a)-APC-Cy7[1:200], CD4 (GK1.5)-PE-Cy7[1:400], Ter-119-PE-Cy7[1:400], F4-80 (BM8)-PE-Cy7[1:400], Sca-1 (D7)-BV605[1:200], B220 (RA3-6B2)-BV421[1:200], and CD138 (281-2)-BV605[1:400] were purchased from BioLegend. ATF-4 (D4B8)[1:1000], XBP1s (E8Y5F) [1:1000], p58IPK (C56E7)[1:1000], ATF-6 (D4Z8V) [1:1000], BiP (C50B12)-PE[1:200], pAKT (S473) (D9E)-PE[1:100] and pS6(S235/236) (D57.2.2E)-PE-Cy7[1:200] were purchased from Cell Signaling Technology. AA4.1-APC[1:100], CD21/35 (8D9)-PE-Cy7[1:200], IgM (11/41)-PerCP-ef710[1:200], and CD8α (53-6.7)-PE and -PE-Cy7[1:400] were purchased from eBioscience (ThermoFisher). CD19 (6D5)-APC-Cy5.5[1:150] and F4-80 (BM8)-PE[1:400] were purchased from Invitrogen. B220 (RA3-6B2)-APC[1:200] was purchased from Tonbo biosciences.

For the initial scRNA-seq experiment was performed using the Chromium Single Cell Gene Expression Solution (10× Genomics), following the manufacturer’s protocol. The MSC was isolated from ten 6-week-old mice (all males). Cells were stained with the anti-mouse antibodies CD31 PE-Cy7, CD45 PE-Cy7 and TER119 PE-Cy7 (Biolegend), Sca-1 PE (eBioscience) and PDGFR-α APC (eBioscience), and 30,000 Lin − Sca-1 + Pdgfr-α + were isolated using a BD Bioscience FACS Aria III Fusion. Cells were washed and resuspended in 250 μl FACS buffer (PBS, 2% FBS, 1 mM EDTA), targeting the required 1000 cells/μl concentration, accounting for a 10–20% loss. We pipetted 9.7 μl cell suspension (concentration of 913 cells/μl, ~ 8800 cells), targeting the recovery of ~ 5000 cells. Single-cell RNA-seq libraries were obtained following the 10× Genomics recommended protocol, using the reagents included in the Chromium Single Cell 3’ v2 Reagent Kit. Libraries were sequenced on the NextSeq 500 v2 (Illumina) instrument using 150 cycles (18 bp barcode + UMI, and 132-bp transcript 3’ end), obtaining ~ 5 × 10⁸ raw reads^{46 (link),47 (link)}.

In experiments assessing general immune cell classes in the blood, approximately 10 million cells were plated in FACS buffer (PBS, 0.2% fetal bovine serum (Sigma), 5 mM EDTA). Prior to staining, the cells were incubated in TruStain FcX amntibody (Biolegend) for at least 5 min at 4°C. A cocktail containing the Live/Dead Fixable Blue stain (Fisher L34962) and antibodies against the following antigens was added to the blocked cells: CD71 PerCP-Cy5.5 (clone RI7217), TER-119 PE-Cy7 (TER-119), TCRγδ PE (UC7-13D5), CD19 Brilliant Violet (BV) 785 (6D5), CD3 BV650 (17A2), CD8 BV510 (53–6.7), Ly6G BV421 (1A8), CD4 Alexa Fluor 700 (GK1.5), Ly6C Alexa Fluor 647 (HK1.4), CD335 FITC (29A1.4) (all from Biolegend); CD11b Alexa 780 (M1/70, eBioscience); CD41 BUV395 (MWReg30, BD Biosciences). All stains were performed for 12–15 min at 4°C. 5 μl of CountBright counting beads (Invitrogen) were added to each samples such that absolute counts per μl of blood could be back calculated. Data were acquired on an LSR Fortessa (BD Biosciences) and analyzed using FlowJo 10.0.8r1 (Tree Star). Significance was calculated by comparing each infected timepoint to values from uninfected mice across infection.