For RNA isolation from the lung, BALB/cJ mice (8-10 weeks old) were obtained from Jackson laboratories (Bar Harbor, ME).  Mice were anesthetized with isoflurane and intratracheally infected with either S. maltophilia, P. aeruginosa, or both (~107 CFU each in 100 µL PBS). Mice were euthanized 24 hours post-infection, and lungs were inflated with RNA-later to preserve RNA integrity. Whole lungs were homogenized, and cells were lysed by bead beating (0.1 mm silica) in Trizol reagent (Invitrogen), and a full lung RNA extraction was performing using a standard protocol (50). Extracted RNA samples were sent to GENEWIZ (South Plainfield, NJ) for DNase treatment, host and bacterial rRNA depletion (Ribo-Zero Gold rRNA Removal Kit (Epidemiology), Illumina), and library preparation using standard protocols. For bacterial RNA-seq, single-end directional samples were DNase treated (DNase I, NEB) using a standard protocol, before being run through a second Trizol extraction to purify the sample of enzyme. Sequencing libraries were constructed using the directional RNA Library Prep Kit (NEB) and tagged for multiplexing via the NE Next Multiplex Oligos for Illumina (NEB). For in vivo bacterial samples, a pathogen-specific probe list for S. maltophilia was generated using previously published methods (28). Briefly, 100 bp probes were generated to cover annotated coding sequences of S. maltophilia, with 15 bp spacer sequences added on either end to allow for amplification of the probe library.   5' SPACER : ATCGCACCAGCGTGT  3' SPACER: CACTGCGGCTCCTCA  Probes completely covered the sense strand of each gene and were also generated to tile every other 100 bp of the antisense strand for a total of 68,704 probe sequences Figure S1). Probes with significant homology to the mouse genome or to bacterial non-coding RNAs (P <0.05 with BLAST analysis) were manually removed from the list. The resulting oligo pool was synthesized by Genscript (Piscataway, NJ). Before hybridization, DNA oligos were amplified and then transcribed (MEGAshortscript T7 Transcription Kit, Ambion), with added biotin-16-UTP (Roche) to generate biotinylated RNA probes.   Prepared cDNA libraries were hybridized to the generated pathogen-specific RNA probes using previously described methods (28) with a few modifications. Briefly, the synthesized probes and the prepared cDNA library were incubated in hybridization buffer for 24 hours at 68°C. Blocking primers were modified for compatibility with NEB multiplexing primers and a 3' ddc' modification was added to maintain barcoding.   Hybridization primer FWD:   AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT/3ddC/  Hybridization primer REV:  CAAGCAGAAGACGGCATACGAGATNNNNNNNNGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT/3ddC/  Hybridized cDNA was isolated via streptavidin beads and then eluted. Enriched samples were amplified using universal primers that maintained sample barcoding before sequencing.