Streptavidin pacblue

Splenocytes were suspended in staining media (SM: 3% FCS, 5 mM EDTA, 0.05% sodium azide, PBS) with the FcR blocking antibody, 2.4G2 and ethidium monoazide (EMA) for live/dead discrimination. Reagents used for staining were either prepared in house: 4-44-biotin, PDCA1 (927)-Alexa 647, CD62L (MEL-14)-Alexa 647; or purchased from Biolegend: CD44 (1M7)-APC/Cy7, CD11c (N418)-PE/Cy7, CD4 (GK1.5)-PE/Cy7, Gr1 (RB6.8C5)-biotin; from BD: CD22.2 (Cy34.1)-PE, TCRβ (H57-597)-PE, I-A/I-E (M5/114.15.2)-PE; from eBioscience: CD11b (MAC-1)-PE, streptavidin-PE/Cy7; or from Invitrogen: streptavidin-PacBlue. Surface stained cells were fixed with 1% PFA, washed and resuspended in SM. For intracellular staining, cells were fixed in 4% PFA in BD Perm/Wash Buffer, washed, blocked (with 5% rat serum), and stained with 4-44-Alexa 647 or GFP-FITC (Rockland) in the BD Perm/Wash Buffer, with a final wash in SM. Cytometry was performed on a LSRII (BD) and data analyzed with FlowJo software.

Harvested bone marrow was stained with BD Pharmingen Biotin Mouse Lineage Panel (cat# 559971, RRID:AB_10053179) and streptavidin-PacBlue (Invitrogen, cat# S11222). Zombie-NIR Fixable Viability Dye (BioLegend cat# 423105) was added to each sample at 1:1000 dilution before sorting alive lineage-negative population (PacBlue-/NIR-). Sorted cells were submitted to VANTAGE for single-cell RNA-seq library prep and processing with 10X Genomics Chromium Controller. Samples were demultiplexed and single-cell gene expression matrices were generated using Cell Ranger software (RRID:SCR_017344). Seurat R package (Butler et al., 2018 (link); RRID:SCR_016341) was used for data analysis (see Supplementary file for R code).