Fastprep lysing matrix b

RNA was isolated as previously described (5 (link)). Briefly, cells were grown to log phase (OD₆₀₀ = 0.5) in the indicated medium. Cells were pelleted and disrupted by mechanical disruption (FastPrep Lysing Matrix B; MP Bio) with 1% β-mercaptoethanol. RNA was isolated with the RNeasy kit (Qiagen), then DNA was removed with Turbo DNase (Invitrogen), and final RNA was concentrated with the RNA cleanup and concentrator kit (Genesee). RNA was reversed transcribed to cDNA with SuperScript reverse transcriptase (Invitrogen). For qRT-PCR, gyrB was used as a housekeeping gene to normalize transcript quantifications, and relative quantification was normalized using the threshold cycle (ΔΔC_T) method. Sequences for tarO, tarG, dltA, fmtA, and gyrB primers can be found in Table S3 in the supplemental material. All qRT-PCR gene expression data were determined from two biological replicates per strain per condition, performed in technical triplicates. For each strain, gene expression was normalized to expression obtained in CA-MHB-Tris, with this value set equal to 1.

DNA isolation from infected mouse footpads were tested with six DNA extraction methods: DNeasy Blood & Tissue Kit (QIAGEN), QIAamp DNA Microbiome Kit (QIAGEN), Maxwell 16 DNA Purification Kit (Promega), PowerSoil DNA Isolation Kit (QIAGEN/Mo Bio), in-house standard phenol-chloroform (Sigma Aldrich) plus FastPrep Lysing Matrix B (MP Biomedicals) and TRIzol (Thermo Fisher Scientific). After assessing the kits on the experimental mouse model, leprosy patient samples were tested with two chosen kits, DNeasy Blood & Tissue and QIAamp DNA Microbiome. All isolation procedures were performed according to the manufacturer’s standard protocols with minor modifications (S1 Text) and DNA was quantified on a NanoDrop D1000.