Quantstudio 6 real time pcr system

Trizol reagent (Invitrogen) was used for total RNA extraction according to the manufacturer’s protocol. RNA was isolated using Direct-zol RNAeasy kits (Zymogen). cDNA was synthesized with BioRadiScript Direct Synthesis kits (BioRad) according to the manufacturer’s protocol. qRT-PCR was performed in triplicate wells using PowerUp SYBR Green Master Mix. Data were analyzed on a QuantStudio 6 Real-Time PCR System (Applied Biosystems).

Yeast strains were grown to an OD of approximately 0.6 to 1 for collection. Three biological replicates were used for each set of experiments (except 2 replicates for RNH1 levels, S1B Fig). Cells were treated with 50 to 100 U of zymolyase in a 1 M sorbitol/100 mM EDTA solution. Cells were lysed and RNA was extracted using the RNeasy kit and RNase-free DNase set (QIAGEN). cDNA was prepared using the Superscript First Strand Synthesis kit (Life Technologies); oligo d(T) primers or locus specific primers (see Table P in S1 Tables) were used for priming during reverse transcription. RT-PCR samples were analyzed using qPCR (performed in technical duplicate with the average shown) with SYBR Premix Ex Taq II (Tli RNase H Plus) (Takara Bio), Power SYBR Green PCR Master Mix (Applied Biosystems), or 2X Universal SYBR Green Fast qPCR (ABclonal) on a QuantStudio 6 real-time PCR system (Applied Biosystems).

The vaginal lavage was diluted tenfold serially with sterile saline and then plated on Columbia blood agar (CBA, Sigma-Aldrich, St. Louis, MO, USA) containing GV-selective supplement (Oxoid, Basingstoke, UK) and 10% horse blood. After incubating the plate under anaerobic conditions at 37 °C for 48 h, the colonies were counted to measure the number of viable GV, and the results were presented as CFU per 1 mL of vaginal fluid. The abundance of GV in the vagina was measured by quantitative polymerase chain reaction (qPCR) analysis of the ratio of GV-specific DNA to the total bacterial DNA in the vaginal fluid (Happel et al., 2020 (link); Naicker et al., 2021 ). After determining the number of viable GV, total DNA was isolated (AllPrep Bacterial DNA Kit, Qiagen, Germantown, MD, USA) from the remaining vaginal lavage and qPCR was performed using the QuantStudio 6 Real-Time PCR System (Applied Biosystems, Foster, CA, USA) with GV-specific primer Ba04646236_s1 (Applied Biosystems) and Pan-bacterial primer Ba04230899_s1 (Applied Biosystems).

To determine stability, RIPK1-KD were made to a final concentration of 1 μg/μl. SYPRO Orange dye was added to the protein to make a final concentration of 2×. UDP-GlcA and UDP-Glc were added in the mix with a final concentration as indicated and incubated at 4 °C for 1 h. The experiments were performed in 384-well plates specific for real-time PCR instrument with a total volume of 20 µl/well. The assay plate was covered with a sheet of optically clear adhesive to seal each well. The assay plate was placed into the Applied Biosystems QuantStudio 6 Real-Time PCR System . The reaction was run from 25 °C, ramping up in increments of 0.05 °C/s to a final temperature of 95 °C with fluorescence detection throughout the experiment to generate a dataset. Data was collected by using QuantStudio 12K Flex software version 1.3 (Applied Biosystems). Melting temperature of the protein (Tm) was determined by performing non-linear fitting of the dataset to a Boltzmann Sigmoidal curve in GraphPad Prism with the following equation: Y = Bottom + (Top − Bottom)/[1 + exp(Tm − X/Slope)], where Y = fluorescence emission in arbitrary units; X = temperature; Bottom = baseline fluorescence at low temperature; Top = maximal fluorescence at top of the dataset; Slope = describes the steepness of the curve, with larger values denoting shallower curves.

Four-week-old human cortical neurons without astrocytes or 14 DIV mouse cortical neurons were used for gene expression analyses. Total RNA was isolated using mirVana kit (Invitrogen) or TRIzol (Invitrogen) according to the manufacturer’s instructions. RNA-seq analysis data are from a previous study^{14 (link)}. For qPCR, a total of 1 μg RNA was used to synthesize cDNA with the SuperScript® III First-Strand Synthesis System (Invitrogen). Quantitative RT-PCR was then performed using SYBR green (Applied Biosystems) and the QuantStudio™ 6 Real-Time PCR System (Applied Biosystems) or the StepOnePlus™ Real-Time PCR System (Applied Biosystems). Expression levels for all genes were normalized to the housekeeping gene GAPDH and expressed relative to the relevant control samples. Primers used for this study are summarized in Supplementary Table 3.