Cfx connect light cycler

Gene expression analysis was done using SYBR Green Supermix (Biorad) in CFX connect light cycler (Biorad), according to the manufacturer’s instructions. Gene-relative expression was calculated using the ΔΔct method [48 (link)] and normalized to a reference control (Rplp0). Primers for qRT-PCR were designed with the assistance of online tools (Primer 3Plus) using at least one exon junction binding site per primer pair where possible. A complete list of primers used is available in the supporting information (S2 Table).

We used qPCR to quantify conserved and unique pregnancy-associated genes in pregnant and non-pregnant cows previously infused with pathogenic bacteria or vehicle medium. Reverse transcription was performed using the Verso cDNA synthesis kit (Thermo Fisher Scientific, Waltham, MA). All primers were designed using the National Center for Biotechnology Information (NCBI) database (S5 Table). Amplification efficiency for each primer pair was evaluated and met MIQE guidelines [23 (link)]. Real time RT-PCR was performed in duplicate 20 μL reactions including forward and reverse primer, iTaq Universal SYBR green master mix (Bio-Rad, Hercules, CA) and 2 to 40 ng cDNA. A CFX Connect light cycler (Bio-Rad) was used with an initial denaturation step at 95°C for 30 sec followed by 40 cycles of a two-step protocol using 95°C for 5 s and annealing and extension at 60°C for 30 s. The primer set for OXTR required a three-step protocol using 63°C as annealing temperature for 5 s and extension at 60°C for 30 s. A no template control was used to determine non-specific amplification for each primer pair. Relative expression for each gene was calculated using the 2^-ΔCt method relative to the geometric mean of the selected housekeeping genes (ACTB, GAPDH, RPL19). Housekeeping gene expression was stable across treatments and pregnancy status (P > 0.05).

Cells were lysed with Buffer RLT Plus (Qiagen) containing 1% beta-mercaptoethanol (Sigma). RNA was purified using the ZR-96-well Quick-RNA kit (Zymo Research). Gene expression was measured using qPCR with reagents from the Power SYBR Green RNA-to-Ct 1-step kit (Applied Biosystems), 50ng RNA per reaction, and 40 cycles on a CFX Connect light cycler (BioRad). qPCR primers are listed in Table S2. Relative expression was calculated using the ΔΔCt method with CTCF as a reference gene and cells where the same dCas9 fusion is targeted to the IL1RN promoter as the controls. Best fit lines were determined using the loess function in R¹⁰⁴ and formula y ∼ x. Half maximal values were calculated using the maximum at any time point. The first time point at which the loess regression reaches a half maximal value is recorded as the time to half maximal. Differentials of the loess regression were also calculated using R to examine how the slope of each trajectory changes over time. Slope analysis was binned into “targeted” and “control” groups to analyze the general effect of dCas9 manipulation at regulatory regions of the respective gene.