Quantstudio 7 flex system

Cells were seeded in 35mm-diameter petri dish with seeding density of 30000 cells per dish and cultured for up to 14 days as described above. RNA was extracted using the RNeasy Plus Mini Kit (Qiagen, 73404) under manufacturer’s instructions at day 1, 7, and 14, and first-strand cDNA synthesized using QuantiTect Rev.Transcription Kit (Qiagen, 205311). Quantitative PCR was performed using the Taqman single-gene expression assay (Thermo Fisher Scientific) in a QuantStudio 7 Flex System (Thermo Fisher Scientific). The following oligonucleotide primers were used: PPARA (Hs00231882_m1) for adipogenesis and RUNX2 (Hs00231692_m1) for osteogenesis. In addition, GAPDH (Hs02758991_g1) was used for normalization. The comparative Ct method was applied to give relative gene expression values. All the values were compared with that gene expression at day 1.

Infectious virus from experiments was quantified using plaque assays performed as previously described using Vero E6-TMPRSS2 cells [30 (link)].
To quantify viral RNA in vitro, 0.2 mL infected culture supernatants were harvested 48 h post-infection and added to 4 volumes of Trizol LS Reagent (Thermo Fisher Scientific). RNA was purified using Direct-zol RNA Miniprep Plus Kits (Zymo, Irvine, CA) according to the manufacturer’s instructions, eluted in 50 μL nuclease-free water, then amplified using iTaq™ Universal SYBR® Green One-Step Kit (Bio-Rad) with QuantStudio™ 7 Flex system (ThermoFisher Scientific). The Ct values of the N gene were normalized to the Ct values of the M-GAPDH for mouse lung tissues or HuGAPDH for HAE cells. Table S1 summarizes the sequences for primer sets.

SALL1 and FOXL2 mRNA levels were quantified by RT-qPCR for two biological replicates each in technical triplicate. RT-qPCRs were performed using 2× Blue S'Green qPCR Kit Separate Rox (Biozym, 331416) according to the manufacturer's instructions with 27.5 ng cDNA and 100 nM of each primer. All experiments were performed on QuantStudio 7 Flex system (Thermo Fisher).
Expression levels were normalized to RPS9 mRNA. The 2-ΔΔCt method was used for analysis of relative SALL1 and FOXL2 expression levels. A one-tailed t-test was applied in these experiments.

Overnight A. baumannii AB5075_UW and resistant clone cultures were grown to mid-log phase (OD₆₀₀ of 0.7) in 20-ml LB cultures prior to RNA extraction. To examine the impact of erythromycin and DHA, AB5075_UW cultures were grown to an OD₆₀₀ of 0.5 in 20-ml LB cultures, which were split into three 4-ml cultures; one culture was treated with 8 μg · ml⁻¹ erythromycin, one culture was treated with 250 μM DHA, and the last culture was treated with both compounds concurrently. Cells were grown for another 30 min before RNA was extracted.
Isolation of RNA was performed using previously described methods (20 (link)). Briefly, cell pellets were lysed with QiaZol (Qiagen). Following phase separation, RNA was extracted from the aqueous phase using the RNeasy purification minikit (Qiagen), incorporating the on-column RNase-free DNase I (Qiagen) treatment according to the manufacturer’s recommendations. Quantitative reverse transcription PCR (qRT-PCR) was performed on a QuantStudio 7 Flex system (Thermo Fisher Scientific) with the Superscript III Platinum SYBR One-Step qRT-PCR kit (Thermo Fisher Scientific). Gene expression was normalized using the constitutively expressed housekeeping gene GAPDH. Oligonucleotides for qRT-PCR are listed in Table S2 in the supplemental material.

Total mRNA was extracted from lung tissue submerged in RNAlater using an RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the protocol from the manufacturer. RNA concentrations were determined using a NanoDrop ND1000 (Saveen Werner, Limhamn, Malmö). Equal amounts of RNA were pooled from several animals in each group (OVA/TH5487 n = 5, OVA n = 4). cDNA was synthesized with an iScript Advanced cDNA Synthesis Kit (Bio-Rad) and mixed with RT² SYBR^® Green ROX™ qPCR Mastermix. A volume of 25 µL of the reaction mixture was added to each well of a RT² Profiler™ PCR Array Mouse Allergy & Asthma PAMM-067ZA plate. The RT-PCR reaction was performed using a QuantStudio™ 7 Flex system (Thermo Fisher Scientific) and data analysis was performed using the manufacturer’s web-based software (https://geneglobe.qiagen.com/analyze). Normalization of gene expression was performed using the following house-keeping genes: B2m, Actb, Gusb, Gapdh and Hsp90ab1.

Total RNA was extracted using an RNeasy Kit (QIAGEN, Valencia, CA) and cDNA was synthesized using Oligo(dT)-primers with a SuperScript First Strand Synthesis kit (Invitrogen, Carlsbad, CA). cDNA was quantified and normalized using a NanoDrop 1000 spectrophotometer. Quantitative PCR was performed in triplicate using Taqman primer/ probe sets (Applied Biosystems) for c-MYC (Hs00905030_m1), SET (Hs00853870_g1), CIP2A (Hs00405413_m1), and for normalization 18S (Hs99999901_s1) genes. Reactions were carried out using 5ng of cDNA and PerfeCta qPCR FastMix (Quanta Biosciences) on a StepOne Real-Time PC System machine (Applied Biosystems). For E2F2, Nucleolin, and 5sRNA RT-PCR reactions were performed with the QuantStudio 7 Flex System (Thermo Fisher Scientific, Grand Island, NY) using Platinum SYBR Green qPCR SuperMix (Invitrogen) according to manufacturer's instructions. ROX reference dye was used to normalize fluorescent signal between reactions. The following primers: Nucleolin forward: 5′-ACTGACCGGGAAACTGGGTC-3′ and reverse: 5′-TG GCCCAGTCCAAGGTAACT-3′, E2F2 forward: 5′-ACA AGGCCAACAAGAGGCTG-3′ and reverse: 5′-TCAGT CCTGTCGGGCACTTC-3′, 5sRNA forward: 5′-GGCC ATACCACCCTGAACGC-3′ and reverse: 5′-CAGCACC CGGTATTCCCAGG-3′, and for normalization GAPDH forward: 5′-TCCTGCACCACCAACTGCTTAG-3′ and reverse: 5′-GGCATGGACTGTGGTCATGAG-3′ were used.