Kapa2g robust hotstart dna polymerase

Total extracted DNA was used for high throughput sequencing (MiSeq platform, Illumina, San Diego, CA, USA) of the bacterial 16S rRNA gene or fungal ITS1 region. We amplified the V3–V4 region of 16S rRNA gene using degenerate barcoded primers 341F (5′-CCTACGGGNGGCWGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′), and the ITS1 region using degenerate fungal primers ITS1-5.8Sfw (5′-CTGTAAAAGTCGTAACAAGGTTTC-3′) and ITS1-5.8Srv (5′-AAGTTCAAAGAYTCGATGATTCAC-3′). PCR amplifications (KAPA 2G Robust Hot Start DNA Polymerase, Kapa Biosystems, Hoffmann-La Roche, Switzerland) were carried out with 25 or 27 cycles, respectively. PCR products were purified and normalized with the SequalPrep™ Normalization Plate Kit (Thermo Fisher Scientific, Waltham, MA, USA). Triplicates of the amplicons were pooled, gel eluted (NucleoSpin gel clean-up, Macherey-Nagel, Düren, Germany), and ligated with sequencing adapters (TruSeq DNA PCR-free LT Sample Preparation Kit, Illumina; KAPA HyperPlus kit, Kapa Biosystems, Hoffmann-La Roche, Switzerland). Amplicon libraries were pooled in equimolar concentrations, validated by the KAPA Library Quantification Kit (Illumina, San Diego, CA, USA), and sequenced on the MiSeq platform using the 2 × 300 bp kit at the CEITEC Genomics Core Facility (CEITEC, Masaryk University, Brno, Czech Republic).

For the library construction, the PCR mixture (10 μl) contained: 1× reaction buffer (2 mM MgCl₂, 0.4 U of KAPA2G Robust HotStart DNA polymerase), 200 μM each dNTP and 2× KAPA2G Robust HotStart ReadyMix (Kapa Biosystems, USA), 1 μM adapter primer, and 2 μl of template, which yielded the first round PCR products. The PCR cycling protocol entailed: 95°C for 3 min; 10 cycles of 94°C for 20 s, 65°C for 2 min, 72°C for 30 s; and 72°C for 5 min. The PCR products were purified using a TIANgel Midi Purification Kit (TIANGEN BIOTECH, Beijing, China).

Total extracted gDNA from earthworm seminal vesicles was used for high throughput sequencing (Miseq platform, Illumina) of the 18S rRNA gene of gregarines in 12 individual earthworms. Two sets of specific primers with barcodes were used in PCR (Table S1 in Supplementary Material). PCR amplifications (KAPA 2G Robust Hot Start DNA Polymerase, Kapa Biosystems) were carried out with 27 cycles. The PCR products were purified and normalized with the SequalPrep™ Normalization Plate Kit (ThermoFisher Scientific). Triplicates of the amplicons were pooled and ligated with sequencing adapters (TruSeq DNA PCR-free LT Sample Preparation Kit, Illumina), pooled in equimolar concentrations, and sequenced. The library was validated by a KAPA Library Quantification Kit (Illumina). The amplicons were sequenced on an Illumina MiSeq using a Miseq Reagent Kit v3 (Illumina).
Data collected from sequencing runs were processed using the Qiime pipeline applying standard procedures such as through quality control and data filtering, clustering analysis, and diversity determination (39 (link)).

3 μl of cDNA amplification mix was added into each snmCAT-seq reverse transcription reaction. Each cDNA amplification reaction containing 1X KAPA 2G Buffer A, 600 nM ISPCR23_2 PCR primer (5′-/5SpC3/AAGCAGUGGUAUCAACGCAGAGU-3′), 0.08U KAPA2G Robust HotStart DNA Polymerase (5 U/μL, Roche KK5517). PCR reactions were performed using a thermocycler with the following conditions: 95°C 3min -> [95°C 15 s -> 60°C 30 s -> 72°C 2min] -> 72°C 5min -> 4°C. The cycling steps were repeated for 12 cycles for snmCAT-seq using H1 or HEK293 whole cells, 15 cycles for snmCAT-seq using H1 or HEK293 nuclei and 14 cycles for snmCAT-seq using human brain tissue nuclei.

Methylated and unmethylated primers specific for the promoter sequences of the target genes, RNF180, DAPK1 and SFRP2, were designed to amplify the bisulfite-modified DNA. The primer sequences used are shown in Table II. The 50-μl reaction mixture contained 2 μl of DNA template, 10 μl of KAPA2G buffer (Kapa Biosystems, Woburn, MA, USA), 1 μl of 10 mM dNTP mix (Kapa Biosystems), 1 μl of each primer at 50 mM, and 0.5 units of KAPA2G^TM Robust Hotstart DNA polymerase (Kapa Biosystems).
The conditions for amplification included a single cycle at 95°C for 5 min and a subsequent 10 cycles at 95°C for 30 sec, melting temperature 8°C (Tm; 0.8°C, descending by 0.8°C for each cycle) for 60 sec and 72°C for 30 sec; then 38 cycles at 95°C for 30 sec, Tm for 60 sec and 72°C for 30 sec; followed by a final extension step of 10 min at 72°C. The PCR products were then electrophoresed on a 2.5% agarose gel and visualized under ultraviolet illumination (ChemiDoc XRS; Bio-Rad, Hercules, CA, USA). Each experiment was repeated at least three times. The operator who performed all assays was blinded to all clinical information.