amplicon sequencing of aerosol sample: medical facility waiting room
Samples were collected after 5 to 18 days of running the AerosolSense for 4 to 8 hours every day (Table 1), after which AerosolSense cartridges were transported to a laboratory for processing. Cartridges were transferred to a 50 ml conical tube with 15 ml PBS and biomaterial was eluted and then concentrated using Ultra-15 10K tubes (Amicon) by centrifuging at 5,000g for 20 minutes. Total RNA was extracted using TRIzol reagent (Thermofisher) following the manufacturers protocol (Pub. No. MAN0001271 Rev. C.0). RNA extract was then DNase treated (NEB) and cleaned using the Monarch RNA Cleanup kit (NEB). For depleting rRNA, we designed sample specific biotinylated RNA probes directly from each sample by generating PCR amplicons of partial 16S (cite 26f 1492r) and 23S rRNA gene sequences (list primers) with T7 promoter sequences appended to the reverse primers. Amplicons were transcribed into biotinylated RNA probes complementary to rRNA sequences using the AmpliScribe T7 Transcription kit (Biosearch) with 30% of the UTP being substituted with biotin-16-UTP (APExBIO). These transcripts were DNase treated, and RNA was cleaned using the Monarch RNA Cleanup kit. These probes were then used for depletion of rRNA in subset of samples. All sequencing libraries were prepared with a stranded template-switching library prep protocol and sequencing was performed on an Illumina iSeq 100 with 150 cycles