Nexseq 500

Total NP-specific CD4⁺ T cells (0.5 × 10⁴ to 3 × 10⁴) were sorted from mLN and lung of PR8-OVA2-infected mice at indicated time points and were provided for library preparation using the 10x Chromium platform. Each sample is pooled from 4 to 12 C57BL/6J mice. Sorting was performed using BD FACSAria III and BDSortAria III. Single-cell capture and cDNA library preparation were performed with the Single Cell 3′ v2 Reagent Kit (10x Genomics) according to the manufacturer’s instructions. Sequencing was performed on one flow cell of an Illumina NexSeq 500 at the Genomics Facility Basel of the ETH Zurich. Paired-end reads were obtained, and their quality was assessed with the FastQC tool (version 0.11.5). The length of the first read was 26 nucleotides (nt), composed of individual cell barcodes of 16 nt, and unique molecular identifiers (UMIs) of 10 nt. The length of the second read, composed of the transcript sequence, was 58 nt. The samples in the different wells were identified using sample barcodes of 8 nt.

CD11b⁺ splenocytes were isolated from wild-type recipient mice 11 weeks after BMT. For hypercholesteraemia experiments, BMT was conducted in Ldlr^−/− mice, four weeks after which a WTD was administered and sustained for seven weeks. Following WTD, mice were killed and CD11b⁺ splenocytes were collected and isolated into TRIzol reagent (Thermo Fisher Scientific). Aortic roots were also removed. RNA was isolated with RNeasy kits (Qiagen) and RNA-seq experiments were conducted as previously described^{37 (link)} on a NexSeq 500 (illumina). Genes were considered differentially expressed if they were significant at 5% false discovery rate (FDR) by DESeq2 and were at least twofold different in average reads per kilobase of transcript per million mapped reads (RPKM). Gene ontology analysis was conducted using the PANTHER database^{38 (link)}. Extended Data Fig. 1 displays RNA-seq data. All data have been deposited into a public repository.

Total RNA from cultured cells was isolated with the QIAGEN RNeasy Mini Kit with on-column DNase digestion. RNA quality checks were performed with an Agilent 2100 Bioanalyzer (Eukaryotic Total RNA Nano kit). Library preparation (500 ng input RNA) was performed with the NEBNext Poly(A) mRNA Magnetic Isolation Module (#E7490) with SPRIselect Beads (Beckman Coulter), the NEBNext Ultra II Single-End RNA Library Prep kit (#7775S), and the NEBNext Multiplex Oligos for Illumina (Index Primers Set 1) according to the manufacturer’s instructions. Library size was confirmed with an Agilent 2100 Bioanalyzer (DNA1000 chip). Pooled libraries were diluted to 1.8 pM (concentrations checked with the Qubit Fluorometer high-sensitivity assay, Thermo Fisher), and sequenced on an Illumina NexSeq 500 instrument with the NexSeq 500 75-cycle high-output kit.
For data analysis, FASTQ files were generated with the bcl2fastq command line program (Illumina). Transcript alignment was performed with Salmon (Patro et al., 2017 (link)). Differential expression analysis (NB4A- vs. DMSO-treated cells) was performed with the DESeq2 R package. DESeq2 “stat” values for each gene were used as inputs to pre-ranked GSEA, where enrichment was tested against the Hallmark gene sets from the Molecular Signatures Database (MSigDB). Access to sequencing data is discussed in the data availability section.