Reverse transcription supermix

Extracted total RNA was reverse transcribed into single-stranded cDNA using a Reverse Transcription Supermix (Bio-Rad, Hercules, CA, USA). Real-time PCR amplification was carried out with SsoFast EvaGreen Supermix (Bio-Rad) using a Bio-Rad Real-Time PCR System under the following conditions: 95 °C for 30 s, 40 cycles of 95 °C for 5 s, and 58 °C for 5 s. All oligonucleotide primers for PCR were chemically synthesized by AIT biotech. Housekeeping genes 18S ribosomal RNA or HPRT1 were used to normalize the expression levels in the subsequent quantitative analyses.

The right lung was placed in 1 mL of Trizol and briefly stored at −20°C. RNA was extracted from each lung following the manufacturer’s protocol. RNA was quantified using a Nanodrop spectrophotometer. cDNA was synthesized from 1 μg of Lung RNA using BioRad Reverse Transcription Supermix for RT-qPCR following the manufacturer’s protocol. Pneumocystis fungal burden was assayed via amplifying a 166-bp fragment of the small ribosomal rRNA (ssRNA) from the mitochondrial genome, as described previously (6 (link), 7 (link)).

Cell cultures were washed with PBS prior to RNA extraction. Total RNA was extracted using RNeasy Miniprep columns (Qiagen) according to the manufacturer’s instructions, including the on-column DNAseI treatment (Qiagen). One μg of total RNA was used for subsequent cDNA synthesis using Reverse Transcription Supermix (Biorad). For qRT-PCR reactions, a total of 20 ng of cDNA was used as a template and combined with primers (see S3 Table), and EvaGreen Supermix (Biorad) and amplicons were generated using standard PCR amplification protocols for 40 cycles on a StepOnePlus Real-Time PCR system (Applied Biosystems). Ct values for each target gene were normalized against Ct values obtained for GAPDH to account for differences in loading (ΔCt). To determine “fold activation” of genes, ΔCt values for target genes were then normalized against ΔCt values for the same target gene for mock-treated cells (ΔΔCt).

To isolate total RNA, the Qiagen RNeasy minikit was used following the RNAprotect bacterial reagent handbook, with modifications. P. aeruginosa biofilms were grown as described above. From the MBEC plate, ∼180 μl of medium was collected from each well and placed into 1.5-ml centrifuge tubes. Collected medium was then centrifuged for 2 min at 12,000 rpm. Pelleted cells were resuspended in 200 μl of RNase free water. Each tube was then centrifuged again for 2 min at 12,000 rpm. The remaining supernatant was discarded. After centrifugation, 30 μl of a 10 mg/ml solution of lysozyme (MP Biomedicals) in 1× Tris-EDTA (Thermo Fisher) was added to each centrifuge tube, followed by incubation at room temperature for 20 min with vortexing every 2 min. The total RNA was then extracted according to the manufacturer’s recommended protocol, including the optional in-column DNase digestion using the RNase-Free DNase set. After RNA extraction, we used a Qiagen DNase Max kit according to the Quick Start protocol provided by the manufacturer. cDNA synthesis was performed using Bio-Rad reverse transcription supermix according to the manufacturer’s recommendations in a Bio-Rad C1000 touch thermal cycler.

High quality total RNA was extracted from frozen liver tissues with the RNeasy Mini Kit (Qiagen) according to the manufacturer’s protocols. Complementary DNA (cDNA) was generated from 1ug RNA by Reverse Transcription Supermix (Bio-Rad #1,708,841). cDNA was then assayed by real-time PCR (RT-PCR) in duplicate using specific primers (Primer sets: ALXR, QT00171514, Qiagen; LGR6, QT00296632, Qiagen) and universal SYBR Green Supermix (Bio-Rad #1,725,121). GAPDH was used as an endogenous control.