Gotaq hot start polymerase kit

cDNA was synthesised with the GoScript reverse transcription system (Promega, UK). Total RNA was reverse transcribed using a 1:1 mixture of random primers and oligo(dT). The GoTaq hot start polymerase kit (Promega, UK) was used to perform standard PCR. PCR conditions were: initial denaturation at 94 °C for 2 min; then 35–40 cycles: 94 °C for 30 s; 58 °C for 30 s; and 72 °C for 30 s followed by a final extension at 72 °C for 5 min. Primer sequences used in standard PCR are shown in Additional file 1: Table S1. PCR products were run on 2% (w/v) agarose gels for 1 h at 95 V and analysed using FluorChem Q software, Alpha Innotech MultiImage III. Optical density peak values were generated with ImageJ software. Excel was used to calculate the percentage splicing index (PSI/ψ) where ψ = exon inclusion/ exon inclusion + exon skipping band intensities.

Bacterial DNA was amplified by PCR using Golay barcoded primers which target the V4 region of 16S rRNA genes (Caporaso et al., 2012 (link)). Template DNA was amplified in triplicate using the GoTaq Hot Start Polymerase kit (Promega, USA). One microliter of template DNA and 1 μL of a unique barcoded reverse primer were added to 48 μL of master mix containing 1x colorless reaction buffer, 1.5 mM MgCl₂, 0.2 mM dNTPs, 0.2 mM forward primer, and 1.25 U of polymerase enzyme. The reaction volumes were placed in a thermocycler and run through the following conditions: 94°C for 3 min (initial denaturation), followed by 35 cycles of 94°C for 45 s (denaturation); 55°C, 40 s (annealing); 72°C, 1.5 min (extension); with a final extension at 72°C for 10 min.

We confirmed the presence of the two c-terminus RUNX2 mutations, initially identified in the CCD patients by “Casa del sollievo e della sofferenza” U.O.C. Genetica Medica—S. Giovanni Rotondo (FG)-Italy, by targeted sanger sequencing of a 470 base-pair (bp) PCR product corresponding to exon 7 and adjacent intron regions of RUNX2 gene (NM_001024630.3). Specific primers (purchased from Thermo Fisher Scientific, Waltham, MA, USA) were designed using Primer3 and BLAST (NCBI, https:www.ncbi.nlm.nih.gov/tools/primer-blast/, accessed on 3 February 2019) and were the following: Forward 5′-TAAGGCCTGAAAGGATGGGGT-3′ and Reverse 5′-ATGTGGGCAAGGGAATGACAA-3′. PCR was conducted with Mastercycler^® ep Gradient S^® (Eppendorf, Milan, Italy) and GoTaq^® Hot Start Polymerase kit (Promega, Madison, WI, USA) by 2 min at 96 °C followed by 35 cycles of 96 °C for 30 s, 60 °C for 30 s and 72 °C for 30 s. A final step of 72 °C for 5 min concluded the PCR program. Purity and identity (by means of length in bp) of PCR product was checked by 1.5% agarose and gel electrophoresis. After purification with FastGene™ kit (Nippon Genetics, Mariaweilerstraße, Düren, Germania), 1.5 µL of the PCR product was used with Dye Terminator Cycle Sequencing (DTCS), Beckman Coulter; Fullerton, CA, USA) Quick Start Kit (Sciex, Milan, Italy) following the manufacturer’s instructions.