Ribozero bacterial kit

For RNA extraction, early-exponential phase cultures, OD_600nm ~0.3, were treated with RNAProtect (Qiagen Inc., CA), and total RNAs were extracted using a hot phenol method as detailed elsewhere (Wen and Burne, 2004 (link); Wen et al., 2006 (link)). To remove all DNA, the purified RNAs were treated with DNase I (Ambion, Inc., TX) and RNA was retrieved with RNeasy Mini kit (Qiagen, Inc.), including an additional on-column DNase I treatment with RNase-free DNase I. The RNA samples were depleted of ribosomal RNA using Illumina's Ribo-Zero Bacterial Kit. 40 ng of RNA were then converted to cDNA, and libraries were generated using Illumina's ScriptSeq v2 Library Preparation Kit. Deep sequencing and post-sequencing analysis were carried out by following our established protocols (Robinson et al., 2010 (link); Lo and Chain, 2014 (link); Love et al., 2014 (link); Maekawa et al., 2014 (link); Kim et al., 2015 (link)).

RNA samples were depleted of rRNA using the RiboZero bacterial kit (Illumina). Libraries of cDNA were prepared using the KAPA stranded total RNA kit (Kapa Biosystems) and sequenced on an Illumina HiSeq 2500. Fastq reads, determined using FastQC v0.11.7 and MultiQC v1.6.dev0, for swarming of P. aeruginosa PAO1 were mapped to its genomic sequence using STAR v2.6.1a. Read counts for individual genes were obtained using HTSeq-count v0.9.1. Significantly differentially expressed genes (adjusted P value ≤ 0.05 and fold change ≥ ±1.5) were identified using DESEQ2 1.20.0 and were then used for further analysis.

Nucleic acids were extracted from collected samples, and DNase was digested twice and checked to be DNA free using PCR. rRNA was removed using the Ribo-Zero bacterial kit (Illumina, San Diego, CA) and evaluated using an RNA HighSens kit (Experion, Hercules, CA). RNA was prepared for multiplexed Illumina RNA-seq (NEBNext Ultra RNA library prep kit for Illumina with NEBNext multiplex oligonucleotides; New England Biolabs, Ipswich, MA) and sequenced on the HiSeqV4 SR100 platform (Campus Science Support Facilities GmbH, Vienna, Austria). Sequence data are available at the European Nucleotide Archive (ENA) under BioProject no. PRJEB13534. Published metatranscriptomic data sets from mice with detectable levels of Mucispirillum were downloaded from the National Center for Biotechnology Information Short Read Archive database and quality filtered using Trimmomatic (89 (link)). Reads were mapped to the M. schaedleri genome using BWA (90 (link)) and analyzed with HTSeq (91 (link)).

Ten milliliters of bacterial culture was collected from triplicate cultures growing exponentially in minimal medium containing glucose (OD at 600 nm [OD₆₀₀] = 0.45 to 0.5) or 10 min after centrifugation and resuspension in minimal medium lacking a carbon source. Cell pellets were immediately frozen on dry ice and stored at −80°C. The mRNA was stabilized by treatment with 10 ml of RNAprotect (Qiagen) (diluted 2 parts RNAprotect to 1 part water) prior to extraction using the RNeasy minikit (Qiagen) with on-column DNase I treatment. The eluates were treated with Turbo DNase (Invitrogen) for 30 min before a subsequent round of purification using the RNeasy minikit. RNA-seq was performed at the Yale Center for Genome Analysis, where the RNA quality was examined using a BioAnalyzer (Agilent) before rRNA was depleted using the RiboZero bacterial kit (Illumina). Approximately 50 million 75-bp paired-end reads per sample were collected using a HiSeq 4000 (Illumina).

Biologically triplicate cultures of uninfected (Δcas6) and chronically infected strains (Δcas6:SSV9.1, Δcas6:SSV11, Δcas6:SSV13, Δcas6:SSV14 and Δcas6:SSV17) were grown to mid-log phase (OD_{600 nm} = 0.20–0.24) in DTU medium. Cells were collected by pelleting and flash frozen in ethanol and dry ice. Total RNA was prepared with a QIAGEN RNeasy kit with on-column DNase treatment with the QIAGEN RNase-free DNase Set according to the manufacturer's protocols. RNA was visualized by agarose gel electrophoresis and quantified with a Qubit 2.0 fluorometer and Qubit RNA BR Assay Kit (Thermo Fisher Scientific, USA). Samples were stored at −80°C before submission to the W. M. Keck Center for Comparative and Functional Genomics at the University of Illinois at Urbana-Champaign (UIUC) for RNA sequencing (RNAseq). rRNA depletion was carried out with the RiboZero bacterial kit (Illumina, USA) and libraries were prepared with the TruSeq Stranded mRNAseq Sample Prep Kit (Illumina, USA). Libraries were quantified by qPCR and sequenced on a NovaSeq600. FastQ files were generated and demultiplexed using bcl2fastq v. 2.20 conversion software (Illumina, USA). Reads were trimmed with Cutadapt and quality filtered using Sickle [43 (link),44 ].