Sh800s sorter

ATAC-seq of dNK cell subsets was performed as previously described^{45 (link)}, with minor modifications. Briefly, dNK1, dNK2, and dNK3 subsets were sorted using the SH800S sorter (Sony). Samples were obtained from a distinct cohort from those were used for the scRNA-seq. Approximately 50k cells were used per library. Samples were lysed in cold lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl₂, and 0.1% NP-40 (Roche) for 3 min on ice to prepare the nuclei. Immediately after cell lysis, nuclei were centrifuged at 500× g for 5 min and the supernatant was discarded. Nuclei extracts were then incubated with the generated Tn5 transposomes and 5× Tris-DMF tagmentation buffer (pH 8.0, 50 mM Tris-HCl, 25 mM MgCl₂, 50% DMF) at 37 °C for 30 min. After DNA purification with a MinElute Kit (Qiagen), PCR was performed to amplify the library for 12–15 cycles according to a quantitative PCR reaction for optimum cycles. The PCR thermocycling program was as follows: 98 °C for 30 s; then 98 °C for 10 s, 63 °C for 30 s, and 72 °C for 1 min for the appropriate number of cycles. Following PCR, sample libraries were purified and sequenced using the Illumina HiSeq X Ten platform with the 150-bp paired-end configuration.

To launch CxNV1 in a naive cell line, Hsu cells were transfected using X-tremeGENE HP DNA transfection reagent (Roche) with plasmid(s) expressing a fluorophore under the control of the Ac5 promoter, and the CxNV1 RdRp, Robin, mutants, or empty under the control of the IE1 promoter with hr5 enhancer, as described above. In initial experiments, both segments were in GFP-expressing vectors; subsequent experiments were performed with Robin in a backbone expressing mCherry instead of GFP, showing that when multiple plasmids were transfected concurrently, >95% of cells received either both or neither plasmid, and yielding no difference in results. As antibiotic-based selection was inefficient, cells were first sorted for successful transfection at 2 to 6 days posttransfection (fluorophore-positive) and then passaged and countersorted for loss of plasmid at 10 to 12 days posttransfection (fluorophore-negative) using a Sony SH800S sorter. At time points from 2 to 9 weeks posttransfection, cells were collected for nucleic acid extraction (including DNase treatment for RNA extractions) and PCR amplification. To determine persistence of viral RNA after loss of plasmid DNA, RT-PCRs were performed with the following primer pairs: oHR672/673 (EF1a), oHR513/514 (CxNV1 RdRp), oHR653/654 (CxNV1 Robin), and oHR691/692 (Neo) (Table S9).

Dissociated cell solution from Nes-dVenus mice were sorted with Beckman Coulter MoFlo Astrios cell sorter or Sony SH800S sorter with 100 μm chips. Cells were first gated with forward scatter (FSC) vs. side scatter (SSC) to remove debris, then gated with DAPI vs. DRAQ5 to select for live cells (DRAQ5⁺/DAPI^-), then gated with GFP 488 vs. FSC to harvest GFP-expressing cells. Cells were collected into DNA LoBind microcentrifuge tubes (Eppendorf #022431021) containing cold oxygenated ACSF supplemented with 1% FBS.

Frozen cells were thawed in a 37°C water bath. Samples from the same group (i.e., stomach region, HPI status) were pooled and washed in warm complete RPMI plus 10% FCS. Cells were then stained with hashtag Abs (B1 for fundus and B2 for antrum) and staining Abs (Supplemental Table 2) for 30 minutes. Cells were extensively washed and sorted on a SH800S sorter (Sony) with a Purity sorting mask. Cells were sorted as single DAPI^–CD45⁺Lin⁺ (T cells), DAPI^–CD45⁺Lin^–CD127⁺ (ILCs) and DAPI^–CD45⁺Lin^–CD127^–HLA-DR⁺ (B cells and myeloid cells). For flow cytometry analysis, samples were stained with the Abs listed in Supplemental Table 3. An LSR Fortessa flow cytometer (BD Biosciences) was used for acquisition, and data were analyzed with FlowJo software (Tree Star).

Human PBMC were thawed and rested overnight. Cells were stimulated for 18 hours by adding aCD3 (UCHT1) and aCD28 (CD28.2) antibodies to the bulk PBMC culture at a concentration of 1 mg/mL and 5 mg/mL, respectively, and CD4⁺ T cells were enriched following stimulation using magnetic negative selection (Stemcell Technologies). Following isolation, T cells were stained with calcein violet live stain (Thermo), Sytox dead stain (Thermo), and aCD45-AF647 (HI30) antibody at 4°C for 30 minutes. After two washes, aliquots of the cells were placed on ice and sorted directly into RLT buffer using a Sony SH800S Sorter for Smart-Seq2 processing and another unstained sample for 10x Chromium analysis. Once the cells were delivered, a third aliquot was loaded onto a Seq-Well array. Single-cell libraries were generated using the Smart-Seq 2, 10x v2, and Seq-Well S³ protocols. For comparison experiments between 10x v3 and Seq-Well S³ human PBMC were thawed and rested overnight. Aliquots of cells were washed, counted and placed on ice prior to processing with 10x v3 and Seq-Well S³ protocols.

CD45⁺ CD11b⁺ F4/80⁺ TAMs were sorted using SH800S sorter (Sony). Cell lysates were prepared in 5 μL buffer TCL (catalog no. 1031576, Qiagen) + 1% β-mercaptoethanol (catalog no. 21985-023, Thermo Fisher Scientific). Samples were sent to Broad Genomic Service, and the SmartSeq2 platform was used to generate RNA-Seq data. Differentially expressed genes were determined using Limma package (51 (link)) in R. GSEA was performed using GSEA software and plotted by ClusterProfiler (52 (link)) and ggplot2 packages in R. RNA-Seq data were analyzed for gene set enrichment in DMBA3-4-TAMs compared with DMSO3-1-TAMs. Original data are available as the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus database (GSE237536).