Kapa sybr fast master mix

For the quantification of 45S and 5S rDNA copy number, quantitative Real Time PCR was performed, as previously described [30 (link)]. Therefore, genomic DNA was isolated from 2-week-old plantlets grown in axenic culture. For qRT-PCR analysis, the LightCycler 480 KAPA SYBR Fast Mastermix (Sigma-Aldrich, Steinheim, Germany) was used in total reaction volumes of 10 μl (5 μl KAPA SYBR Fast Mastermix, 0.5 μl of each primer [10 μM], 0.5 ng gDNA, filled up with distilled water). The analysis was performed using a Light Cycler 480 (Roche Diagnostics, Mannheim, Germany). Data was normalized by amplification of the Ubiquitin 10 gene. For primer sequences see S3 Table.

DNA was isolated from fecal pellets using the QIAamp DNA stool mini kit (Qiagen) according to manufacturer’s instructions for pathogen detection. Copy number of total bacterial 16S rRNA was determined by qPCR using the oligonucleotides 5′-GTG CCA GCM GCC GCG GTA A-3’ (515f) and 5′-GAC TAC CAG GGT ATC TAA T-3’ (805r) (Wasimuddin et al., 2020 (link)) mixed with KAPA SYBR Fast master mix (Sigma) and amplified with a CFX95 Touch™ Real-Time system (BioRad). Conditions were 50 cycles at 95°C for 30°s and 56°C for 50 s after an initial denaturation at 95°C for 3 min. The total reaction volume of 23 µL contained 2 µL of spermine (Sigma) dissolved in ddH₂O at a concentration of 0.16 g L⁻¹ to overcome polymerase inhibition by DSS (Krych et al., 2018 (link)).

Hearts were collected on days 2, 4 and 7 after injury, and total RNA was extracted using TRIzol Reagent (Sigma-Aldrich, T9424) and chloroform (AppliChem). For each specimen 500 ng of total RNA was reversed transcribed into cDNA using PrimeScript RT reagent kit (TaKaRa RR037a) and oligo dT primers according to the manufacturer’s protocol. Prior to cDNA synthesis, samples were subjected to TURBO^TM DNase (Invitrogen) treatment for 1 h at 37 °C and 10 min at 75 °C. Quantitate PCR was conducted using KAPA SYBR FAST Master Mix (Sigma-Aldrich, KK4611) on a Roche Lightcycler 96 (Roche Life Science). Cycling conditions were as follows: 2 min at 50 °C and 10 min at 95 °C (Pre-incubation) followed by two-step PCR for 40 cycles of 15 s at 95 °C and 60 s at 60 °C. Expression levels were calculated using the comparative CT method and calculated 2^−ΔΔCt values are presented. Values for specific genes were normalized to GAPDH as a constitutively expressed internal control. Primers used are;
eYFP: F: 5′-ACGTAAACGGCCACAAGTTC-3′ R: 5′-AAGTCGTGCTGCTTCATGTG-3′
Tbx5: F: 5′-CTCCCAGCAAGTCTCCATCA-3′ R: 5′-GGCCAGTCACCTTCACTTTG-3′
Gapdh: F: 5′-AGGTCGGTGTGAACGGATTTG-3′ R: 5′-TGTAGACCATGTAGTTGAGGTCA-3′

RNA extraction was performed for each sample using the NucleoSpin RNA kit (Macherey-Nagel). Total RNAs were quantified using a Nanodrop instrument (ThermoFisher). For sequencing, all samples were sent to the GIGA genomics platform in Liège, Belgium. Genome quality was assessed using the RNA 6000 Nano Chip kit on a 2100 bioanalyzer (Agilent). cDNA libraries were prepared by employing the universal prokaryotic transcriptome sequencing (RNA-seq), prokaryotic AnyDeplete kit (Nugen) according to the manufacturer’s instructions. cDNA libraries were quantified and normalized by using the Kapa SYBR fast mastermix (Sigma-Aldrich) with P5-P7 Illumina primers according to the manufacturer’s instructions. Prepared libraries were sequenced on a NextSeq 550 device (Illumina) by using the following parameters: paired end, 80 cycles read 1, 8 cycles index, and 80 cycles read 2.