Novaseq 150

Total extracted RNA quality was confirmed for sequencing using the Agilent 2100 Bioanalyzer system using the RNA 6000 Pico Kit (Agilent). Bacterial and mammalian rRNA depletion, and Illumina library preparation for strand-specific RNA-sequencing, was performed by Genewiz, then samples were sequenced on an Illumina NovaSeq 150 bp paired-end run.

HTGTS was performed using the linear amplification-mediated (LAM) platform^{31 (link)} with modifications as described^{31 (link)} to quantify both single DSB rejoining as well as translocation to other DSBs (JoinT-seq). Briefly, 10 μg of isolated gDNA was used to prepare amplicons for Illumina Nova-seq 150 bp paired end sequencing. Sequence reads were aligned to the hg38 genome build and normalized to 2,914,360 and 793,219 each for IGHM-6 and IGHM-1 bait analyses, respectively. Hotspot determination used MACS2 with an FDR-adjusted P-value cutoff of 10^{− 9} as described.^{32 (link)}

Genomic DNAs of Arthrobacter globiformis wild type and spontaneous resistance mutants (K, M, O, P, Q, R, Y, and BB) were extracted by using a Qiagen DNeasy blood and tissue kit with optimized cell lysis for Gram-positive bacteria. DNA quality was confirmed by using gel electrophoresis, a NanoDrop One UV-Vis spectrophotometer, and a Qubit fluorometer (Invitrogen Qubit DNA-HS assay kit, catalog no. Q32851). Library preparation and sequencing were performed by UCB QB3 with Illumina NovaSeq 150-bp, paired-end reads. SNPs and indels were identified in mutants and the wild type by aligning raw, paired-end reads to the A. globiformis reference genome NBRC12137 (NCBI accession NZ_BAEG01000085) individually using Snippy (version 4.5.0; https://github.com/tseemann/snippy) with standard parameters. Snippy analyses were run on the Galaxy (99 (link)) computing cluster. SNPs/indels with 150 raw reads or fewer were excluded from the analysis.