Nextseq sequencer

Mock-infected and TB40/E-5–infected mixed PBMCs from the in vitro model were stained with calcein-violet AM, CD3/19/20/56^APC, and CD14^AF700, and calcein⁺ and CD3/19/20/56⁻/CD14⁺ cells were sorted on the BD Fusion. Isolated cells were centrifuged at 350g for 10 min at 4°C and then resuspended in 0.1% BSA/PBS to a final concentration of 3000 cells/μl and vortexed to ensure a single-cell suspension. Trypan blue staining was done to confirm that cells entering the scRNA-Seq workflow had greater than 95% viability. For mock- and CMV-infected monocytes isolated from in vitro infections, single cells were then encapsulated into oil droplets using the Bio-Rad ddSEQ Single-Cell Isolator. The Illumina Bio-Rad SureCell WTA 3′ Library Prep kit PBMC protocol was used to generate the cDNA libraries from RNA released from encapsulated cells during cell lysis and oil droplet disruption. Final libraries were quantitated and evaluated for quality control on the Agilent bioanalyzer in the CIID and MGH. scRNA-Seq libraries from calcein⁺CD45⁺14⁺ monocytes obtained from cardiac transplant recipients were constructed using the inDrops-Seq methodology in conjunction with the Harvard Medical School Single Cell Core as previously described (65 (link)). All scRNA-Seq libraries were sequenced on a Next-Seq Illumina sequencer at the Molecular Biology Core Facilities at Dana-Farber Cancer Institute.

RNA samples were sequenced at Biomedicum Functional Genomics Unit (FuGU) with Illumina NextSeq sequencer (Illumina, San Diego, CA, United States) in High output run using NEBNext^® Ultra^TM II Directional RNA Library Prep Kit for Illumina. The sequencing was performed as single-end sequencing for read length 75 bp. The count data was used to calculate differential expression statistics with the DESeq2 software in the R environment. Genes with an adjusted p-value for the log2-fold change <0.05 were considered significant. Gene Ontology (GO) analysis was performed using DAVID Bioinformatics Resources 6.8.

To confirm the correct taxonomic assignment of the A. radioresistens strains included in this study, we performed Whole Genome Sequencing (WGS) using Illumina technology. Nextera XT DNA libraries (Nextera XT DNA Library Prep Kit, Illumina, San Diego, CA, USA) were generated from genomic DNA and sequenced on an Illumina NextSeq sequencer (Illumina, San Diego, CA, USA) followed by short-read genome assemblies using SPAdes 3.12 [23 (link)] (Center for Algorithmic Biothechnology, St. Petersburg, Russia). All genome sequences were deposited at NCBI GenBank, BioProject ID PRJNA224116, Acc. Nos. JAATOZ000000000-JAATPL000000000 and JANRFO000000000-JANRFV000000000 (Table 3). In silico analyses were performed using the EZ Biocloud ANI calculator (https://www.ezbiocloud.net/tools/ani, 22 June 2022) and the DSMZ type strain genome server (TYGS, https://tygs.dsmz.de, 29 May 2021) to determine digital DNA-DNA hybridization values [24 (link),25 (link)]. For taxonomic comparisons, our isolates were compared to the type strain genome A. radioresistens NBRC 102413^T (GenBank Acc. Nos. AP019740-1). For the determination of a species affiliation of a strain, a cut-off of 94–96% is given for the ANI and a value of 70% for the dDDH [21 (link)].

Total RNA was isolated using an RNeasy mini kit (Exiqon; Qiagen, Hilden, Germany) following the manufacturer’s protocol. RNA quality and quantity were analyzed on ND-1000 spectrophotometer (Nanodrop Technology, USA). Furthermore, RNA quality was assessed by Bioanalyser RNA 6000 kit (Agilent Technologies, CA, USA) to determine the RIN number. Only samples with RIN ≥ 7 were included for sequencing. Next-generation sequencing (NGS) libraries were constructed with the TruSeq® Stranded mRNA LT kit (Illumina, San Diego, CA) according to the manufacturer’s protocol. Bioanalyser (Agilent Technologies, CA, USA) was used for quality control of indicated steps as recommended by the manufacturer. Single-read sequencing for 75 cycles was run in an Illumina NextSeq sequencer (Illumina Inc., San Diego, USA) using the NextSeq 500 High output kit (Illumina Inc., San Diego, USA).