Scriptseq complete kit for bacteria

An overnight culture of S. aureus in TSB was diluted 100 times in a fresh TSB (2 mL in a 15 mL test tube) and incubated in a shaking incubator at 37 °C for 2 h. Then 1 μg/mL of streptozotocin or floxuridine were added to the culture and further incubated for 3 h. After immediate stabilization of RNA in all samples by RNAprotect Bacteria Reagent (Qiagen), total RNA was isolated with RNeasy Mini Kit (Qiagen) according to the manufacturer’s recommendations. For each condition, three RNA samples were isolated from three independent bacterial cultures. The isolated RNAs were sent to the Center for Genomics and Bioinformatics at Indiana University. Sequencing libraries were constructed using the ScriptSeq Complete Kit for Bacteria (Epicentre). The statistical analysis of the RNA-seq results was done with DeSeq. 2 as described before^{47 (link)}.

Total RNA was extracted from the stored RNAprotect solution using an RNAeasy Qiagen kit (Qiagen, Valencia, CA) as indicated by the manufacturer’s instructions, followed by an additional treatment with DNase I (Qiagen, Valencia, CA) to remove traces of DNA. rRNA depletion and mRNA enrichment were performed using an Illumina Ribo-Zero rRNA removal kit (catalog no. MRZ116C) according to the instructions of the manufacturer (Illumina, San Diego, CA). RNA concentrations were measured using an RNA Qubit assay (Invitrogen, Burlington, Ontario, Canada), and RNA integrity was evaluated using a Bioanalyzer RNA Pico assay (Agilent Technologies, Santa Clara, CA) (18 (link)). cDNA libraries were constructed using a ScriptSeq Complete kit for bacteria (Epicentre, Madison, WI). cDNA was then purified using an Agencourt AMPure XP system (Beckman Coulter, Beverly, MA), and the second cDNA was generated by adding the Illumina adapters as the forward primer and a ScriptSeq index primer as the reverse primer. The resulting libraries were purified using an AMPure XP system (Beckman Coulter, Beverly, MA), quantified with the DNA Qubit assay, and evaluated using the Bioanalyzer sensitivity DNA assay (Agilent Technologies, Santa Clara, CA). One hundred single reads were generated using the Illumina HiSeq 2000 platform at the National Center for Genome Research in Santa Fe, NM.

Cells were grown in RPMI to exponential growth phase; then ZnSO₄ (20 μM), CP (1.1 μM), or the mix of ZnSO₄ (20 μM) and CP (1.1 μM) was added to the culture. After 4 h incubation of the culture at 37°C, total bacterial RNA was isolated using the RNeasy minikit (Qiagen) with optional on-column DNA digestion according to the manufacturer’s instructions. After purification, contaminating DNA was removed with RNase-free DNase I. RNA was then purified again using RNeasy Mini columns. The purified RNA was sent to the Center for Genomics and Bioinformatics at Indiana University. Sequencing libraries were constructed using the ScriptSeq Complete Kit for Bacteria (Epicentre).