RNA isolation and RNA-seq library preparation.

Dedicated lots of reagents and tips were used for RNA extraction of all new specimens. RNA was isolated from bacterial cultures and biopsy specimens the day after collection using the Qiagen fibrous tissue kit with the modifications described in reference 31. RNA quantity and integrity were assessed with a BioAnalyzer (Agilent).

Reagents from the Qiagen FastSelect for bacteria and for human kits were combined to deplete rRNAs from both species from the 20 RNA specimens (4 mid-log-phase cultures, 8 pustules, and 8 wounds) obtained from the 8 volunteers who were infected specifically for this study per the manufacturer’s instructions. RNA-seq libraries were prepared with a Roche KAPA kit. Libraries were pooled to account for the extra depth needed to see bacterial transcripts in the pustules. For the pilot study (31) and this study, our goal was to obtain 400 million reads from pustules, 100 million reads from wounds, and 50 million reads from mid-log cultures. Libraries were sequenced on a NovaSeq 6000 (Illumina) at the Center for Medical Genomics at Indiana University School of Medicine. Paired-end 150-bp reads were obtained. The actual numbers of reads for the new samples are presented in Table 1.

The reads obtained from the 4 volunteers who participated in the pilot study were obtained following similar procedures except the Ribo-Zero Gold rRNA removal kit (Epidemiology) (Epicentre Biotechnologies) was used to deplete human and bacterial rRNAs, libraries were constructed using the TruSeq stranded total RNA library kit (Illumina), and paired-end 75-bp reads were obtained on a Hi-Seq 4000 (Illumina).