IFN treatment of immune cell populations
Frozen Ficoll purified PBMC from five healthy subjects were acquired from Bloodworks Northwest. After thawing, CD14 monocytes, T, B, and NK cells were enriched by STEMCELL technologies magnetic negative selection kits. T cells were further depleted from non-T cell populations using the STEMCELL EasySep CD3 positive selection kit.
Three to eight hundred thousand cells from each immune cell subset or total PBMCs were seeded in 200 μL of RPMI-1640 media with 10% human AB serum per well in a 96-well plate. Cells were treated with IFN-α2A (1000 units/mL, PBL Assay Science), IFN-β (100 units/mL), IFN-γ (25 ng/mL, Biolegend), IFN-λ1 (100 ng/mL) or were left unstimulated for 21 hours in 37°C.
scRNA-seq of treated populations
After treatment, cells were washed and prepared for 10x Genomics Flex scRNA-seq. Cells were processed according to the 10x Genomics protocol for Fixation of Cells and Nuclei (CG000478, Rev C), with volumes scaled from 1 mL to 250 μL for plate-based sample preparation. Samples were fixed in a final concentration of 4% Paraformaldehyde for 1 hour at 25°C, then quenched and washed with 200 μL of PBS + 0.02% BSA (Costar) before barcoding for multiplexing using 10X Genomics Fixed RNA Feature Barcode Multiplexing Kit (#PN-1000628).
Probe hybridization was completed according to the 10x Genomics Flex Chromium User Guide (CG000673, Rev A). After 24 hours of probe hybridization at 42°C, samples were diluted in Post-Hyb Wash buffer and 11 µL was removed for counting. The counting fraction was stained 1:5 with 44 μL of PI solution and counted on a Nexcelom Cellaca MX cell counter. Samples were then pooled at equivalent cell concentrations at a minimum of 50,000 cells/sample and were washed by incubation in Post-Hyb Wash Buffer for 3 rounds of 10 minutes at 42°C, then strained through a 30 μm filter (Miltenyi).
The single cell suspension pool was loaded onto a 10x Genomics Chromium Chip Q for GEM generation at 400,000 cells per chip well. GEM recovery, pre-amplification, and library generation were performed as described int he Flex User Guide. Final scRNA-seq libraries were sequenced using an Illumina NovaSeq X 25B PE300, Novaseq S2 PE200, or a NovaSeq S4 PE100 flow cell, targeting a minimum read depth of 10,000 reads per cell.