Nextseq 2000 platform

ChIP and ChIP-Rx (ChIP-sequencing) with spike-in normalization were performed as previously described^{22 (link)}, with the slight modification of using sonication (20 min, 25% amplitude, 10 s on, 30 s off) to fragment the DNA prior to immunoprecipitation. CUT&RUN followed by sequencing was based on ref. ^{70 (link)} When specified, 10% U2OS cells were added as spike-in. The following primary antibodies were used for ChIP (3 µg) or ChIP-sequencing (15 µg): anti-RNAPII (Santa Cruz Biotechnology Cat# sc-17798, RRID:AB_677355), anti-SPT6 (Novus Cat# NB100-2582, RRID:AB_2196402, 1.5 µg for ChIP and 10 µg for ChIP-sequencing), anti-SPT5 (Santa Cruz Biotechnology Cat# sc-133217, RRID:AB_2196394), anti-RTF1 (Bethyl Cat# A300-179A, RRID:AB_2185963), anti-RNAPII phosphor-Ser2 (Abcam Cat# ab5095, RRID:AB_304749), anti- RNAPII phosphor-Ser5 (BioLegend Cat# 904001, RRID:AB_2565036), anti-SPT4 (Cell Signaling Technology Cat# 64828, RRID:AB_2756442, 1.5 µg for ChIP), anti-CTR9 (Bethyl Cat# A301-395A, RRID:AB_960973). All libraries were sequenced on the NextSeq2000 Illumina platform, paired-end sequencing with 60 cycles for read1 and 60 cycles for read2, except for BLISS libraries that were sequenced on NextSeq2000, single-read sequencing with 101 cycles for read1.

Library preparation, short- and long-read sequencing (Illumina and Oxford Nanopore technologies, respectively), and de novo assembly were performed by the Microbial Genome Sequencing Center (MiGS; Pittsburgh, PA, USA). For DMS3 and JG024 phage reboot clone and PA14 and PAO1 host defense system deletion verification, Illumina NextSeq 2000 sequencing was performed, presented in Table S5. Illumina paired-end reads (2 × 151 bp) were obtained using the Illumina DNA Prep Kit, IDT 10 bp UDI indices, and the Illumina NextSeq 2000 platform (80 (link)). Demultiplexing, quality control, and adapter trimming were performed by MiGS with bcl-convert (v4.0.3). Quality control was checked using FastQC (v0.11.5) (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and MultiQC (v1.11).
ONT sequencing libraries for JG024 WT were prepared using Oxford Nanopore’s “Genomic DNA by Ligation” kit (Oxford Nanopore Technologies, Oxford, UK) and sequenced on a MinION R9 flow cell. Base calling for ONT long reads was performed using Guppy HAC basecalling mode (v4.2.2) (81 (link)). bcl2fastq v2.20.0.445 (82 ) and Porechop v0.2.3_seqan2.1.1 (83 (link)) were used for quality control and adapter trimming for Illumina and ONT sequencing, respectively.

Four independent biological replicates of C. difficile CD2015 were grown in BHI medium supplemented with 25 µM UroA or vehicle (DMSO) for 24 h. Following growth, cells were pelleted, supernatant was removed, 1 mL of RNA later was added, and the pellet was stored at −80°C until needed. Total RNA was extracted using the RNeasy kit (Qiagen), and residual DNA was removed using the TURBO DNA-free Kit (Invitrogen). RNA-Seq was performed at SeqCoast Genomics. RNA samples were subjected to ribosome depletion and sequenced on an Illumina NextSeq 2000 platform using a 300-cycle flow cell kit to produce 2 × 150-bp paired reads. After demultiplexing, read trimming, and FastQC analysis, transcript expression was determined using Salmon in Python 3.10 and subsequent analysis with DESeq2 and apeglm in R v4.3.0 (63 – (link)66 (link)).

L. monocytogenes isolates for whole-genome sequencing were grown overnight (16–18 hours) in BHI. Genomic DNA was purified using the MasterPure Gram-positive DNA purification kit (Epicentre) per the manufacturer’s instructions, except that 5 U/μl mutanolysin was used instead of lysozyme. DNA was submitted to the Microbial Genome Sequencing Center (MiGS, Pittsburgh, PA) for whole-genome sequencing using the Illumina NextSeq 2000 platform (150 bp paired-end reads). Reads were mapped onto the L. monocytogenes 10403S reference sequence (RefSeq accession number NC_017544) using Bowtie2 (version 2.3.5.1) [82 (link)]. Single nucleotide polymorphisms (SNPs) were called using SAMtools and BCFtools (version 1.9) [83 (link),84 (link)].

Whole-genome sequencing was performed at the NIID, Japan. The whole-genome sequences of SARS-CoV-2 were obtained using the PrimalSeq protocol to enrich the cDNA of the SARS-CoV-2 genome using multiplex RT-PCR amplicons with a multiplexed PCR primer set, as proposed by the Welcome Trust ARTIC Network. We used the primer for multiplex PCR amplification selected by Itokawa et al. [19 (link)]. The PCR products from the same clinical sample were pooled, purified, and subjected to Illumina library construction using the QIAseq FX DNA Library Kit (QIAGEN, Hilden, Germany). The Illumina NextSeq 2000 platform (Illumina, San Diego, CA, USA) was used to sequence the indexed libraries. Detailed methods for next-generation sequencing are reported in the literature [19 (link)].