Nextseq 550 platform

For RNA sequencing, RNA quality was assessed with TapeStation (Agilent, Cernusco sul Naviglio, Italy), and only RNA with RIN > 8 was used for library production. According to the manufacturer instructions, indexed libraries were prepared from 1 µg of purified RNA using TruSeq Stranded Total RNA Sample Prep Kit (Illumina Inc., Berlin, Germany). Libraries were pooled and sequenced (paired-end, 2 x 100 bp) on NextSeq 550 platform (Illumina Inc., Berlin, Germany). A Ribo-Zero Gold rRNA Removal Kit specific for yeasts was used (Illumina Inc., Berlin, Germany) to remove rRNA. Libraries were pooled and sequenced (paired-end, 2 x 75 cycles) on NextSeq 550 platform (Illumina Inc., Berlin, Germany). Three independent biological replicates were performed.

To assess PBMCs vitality, cells were stained with viability dye (Zombie NIR; Biolegend) for 15 min at RT and Fc-block was performed with 1% human serum for 10 min at RT. PBMCs were then stained for 15 min at RT with fluorophore-conjugated antibodies listed in Table S1. Finally, cells were washed in 2% fetal bovine serum/PBS and live CD45^pos/HLA-DR^pos/lineage^neg (CD3, CD19, CD56) were immediately FACS sorted on a FACSAria III (BD Biosciences) (Figure S1A). Cells resuspended in 0.5 ml PBS 1X plus 0.04% BSA were washed once by centrifugation and counted with an automatic cell counter (ThermoFisher; Countess II). About 20,000 cells per sample were loaded into one channel of the Chromium Chip B using the Chromium Single Cell 3’ Reagent Kits (v3 Chemistry) (10X Genomics). 50 ng per sample of the barcoded and amplified cDNA was used then for constructing the sequencing libraries. Sequencing was performed on the NextSeq550 Illumina Platform, generating on average about 477 million reads per sample (on average about 67,000 reads per single cell recovered) following the configuration of the sequencing RUN indicated by the Single Cell 3’ scRNAseq v3 protocol.

After RNA extraction, a RNA quality control was performed with the 4200 Tape Station system (Agilent). Only RNAs having an RNA Integrity Number (RIN) greater than 6 were used for library preparations. Total-RNA-Seq library preparation was performed starting from 0.5 ng of total-RNA with the SMART-Seq Stranded Kit (Clontech-Takara). Libraries obtained were qualitatively assessed by using TapeStation 4200 (Agilent) and quantified by Qubit Fluorimeter (Thermo Fisher Scientific). Afterward, they were multiplexed in equimolar pools and sequenced on a NextSeq-550 Illumina Platform generating at least 60 million 75bp-paired-end reads per sample.
RNA-Seq data are available at GEO under accession number GSE160362.