Quanta cdna synthesis kit

Samples for analysis of PaNAC03 expression levels during embryo development, was a generous gift from Drs. Irena Molina and Malin Abrahamsson. Briefly, samples were collected from five sequential developmental stages (classification based on Zhu et al. [35 (link)]): +PGR (Proliferating cultures + Plant growth regulators (PGR) five days after subculture), —PGR (Proliferating cultures —PGR five days after subculture), EE (Early embryos differentiated after one week on maturation medium); LE1 and LE2 (late early embryos developed after two and three weeks on maturation medium, respectively). Three independent samples were collected for every stage and frozen in liquid nitrogen and stored at −80 °C until extraction. Total RNA were extracted with the Spectrum Plant Total RNA kit (Sigma Aldrich) after DNAse I treatment one μg of total RNA was reverse transcribed with the Quanta cDNA synthesis kit (Quanta Biosciences).

hCMEC cells were cultured and seeded in 24-well plates as described above. Plates (24 wells) were incubated for 4 days at 37°C and 5% CO₂. Thirty minutes before infection, media was exchanged for fresh media. Mid-exponential growing COH1 and COH1∆iagB were prepared as described above, and hCMEC cells were infected at an MOI of ~20. Infection was performed for 5 hours at 37°C and 5% CO₂. Cells from each well were collected and RNA extracted using the NucleoSpin RNA purification kit (Macherey-Nagel) per manufacturer’s protocol. cDNA was generated using the Quanta cDNA synthesis kit (Quanta Biosciences), and transcript abundance was determined using PerfeCTa SYBR Green reagent. Fold changes in transcript abundance were calculated using ∆∆CT, by which target gene transcript levels were normalized to those of housekeeping gene, Gapdh. Quantitative real-time (qRT) PCR data represent the average of three biological replicates with three technical replicates each. qPCR primers used in this study are listed in table S2.

Vaginal lavage fluid was collected as described in reference 38 (link) and filtered through 0.22-μm Spin-X centrifuge tube filters (Costar) to remove contaminants. Triplicate log-phase cultures of USA300 were pelleted and resuspended in filtered lavage fluid. Following a 2-h incubation at 37°C, bacteria were collected by centrifugation, resuspended in TRIzol, and lysed by bead beating, and RNA was isolated using the Direct-zol RNA MiniPrep Plus kit, as described above. RNA was treated with Turbo DNase (Invitrogen) to remove contaminating DNA. cDNA was generated using the Quanta cDNA synthesis kit (Quanta Biosciences), and qPCR was performed using PerfeCTa SYBR green reagent (Quanta) and a CFX96 real-time PCR thermal cycler (Bio-Rad). Fold changes were calculated using the Livak method (116 (link)).