Cfx real time qpcr instrument

Larvae from heterozygous in-crosses (dmist^i8/+ or dmist^vir/+) were genotyped by tail biopsy at 3 dpf (Wilkinson et al., 2013 (link)) and allowed to recover fully in individual wells of a square welled 96-well plate before euthanising at 5 dpf. RNA was extracted from three 5 dpf embryos of each genotype by snap freezing in liquid nitrogen and TRIzol RNA extraction (Ambion 15596026) with the following modifications to the manufacturer’s protocol: 400 μl total TRIzol reagent used to homogenise larvae using a pellet pestle homogeniser, and 5 μg glycogen (Invitrogen, Cat# 10814010; 20 μg/μl) was added to the RNA solution after chloroform extraction to aid precipitation of the RNA. The cDNA library was synthesised from high-quality RNA (Agilent AffinityScript qPCR cDNA synthesis kit 600559), diluted 1:10, and gene-specific primers (Table 2) were used for amplification of target genes with SYBR green mastermix in a Bio-Rad CFX Real-Time qPCR instrument. Gene expression levels were normalised to the housekeeping gene ef1alpha (primers in Table 2) and analysed using custom MATLAB scripts (MATLAB v9.2 2017, The MathWorks 2017).

qPCR was used to measure the abundance of B.thetaiotamicron in CD19^−/− mice colonized with isogenic B.theta strains (see description below). 40 days post-colonization, fecal pellets were collected, snap frozen in liquid nitrogen, and stored at −80 °C until use. Fecal DNA was extracted using the Allprep DNA/RNA Isolation Kit (with a 3 min bead beating step) (Qiagen). Sample DNA concentration was measured using a Nanodrop Spectrophotometer. Total bacteria were estimated using universal 16S qPCR primers (338F/518R), and B.theta abundance was estimated using B.theta-specific primers (Supplementary Fig. S13). Ct values were converted into amplicon copies using the following equation 10^{((Ct-35)/−3)}. Amplicon copies were standardized to the amount (nanograms) of DNA added to each reaction.  qPCR was performed on a CFX Real-Time qPCR Instrument (Bio-Rad) using the iTaq Universal SYBR Green Supermix (Bio-Rad). Primer sets used in qPCR experiments are provided in Supplementary Table 2.