High fidelity restriction endonucleases

The chemicals used for DNA modifications, sulfosuccinimidyl 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)−2000] (Sulfo-PEG₂₀₀₀-DSEP) was from Nanocs Lipid. Sulfosuccinimidyl 6-(4,4′-azipentanamido)hexanoate (Sulfo-LC-SDA), 0.4% trypan blue, propidium iodide (PI), calcine-AM, cell culture medium (Dulbecco’s Modified Eagle Medium, DMEM, Gibco 21063), and a live/dead bacterial viability kit were purchased form Thermo Scientific. Klenow DNA polymerase and DNA modification enzymes were from New England Biolabs (NEB), including S1 nuclease, high-fidelity restriction endonucleases (EcoRI-HF, HindIII-HF, and PstI-HF), micrococcal nuclease, and corresponding buffers. Other chemicals and reagents were all analytical grade without further purification. The DNA oligonucleotides used in this work were listed in Supplementary Table 2. Unmodified DNA oligonucleotides were basically synthesized by Sangon Biotech (Shanghai) Co., Ltd.

SNPs within CRP, IL-6, VDR, eNOS, and COL1A1 genes were carefully selected based on the following criteria: (i) polymorphic with minor allele frequency >5%, (ii) confirmed functional SNP influencing mRNA expression, (iii) significant participant in inflammatory conditions evidenced by published reports, and (iv) validated at NCBI’s refSNP cluster (http://www.ncbi.nlm.nih.gov/snp (accessed on 20 January 2021). Twenty SNPs (Table 1) within these genes were chosen and genotyped. DNA was extracted from the whole blood using the salting-out method. A total of 25 µL of reaction mixture was used to amplify the extracted DNA by employing the polymerase chain reaction (PCR). Amplified DNA was digested with respective high fidelity restriction endonucleases (New England BioLabs Inc., Ipswich, MA, USA). The genotypes were scored and visualized with 2–3% agarose gel electrophoresis depending upon the size of the product. All the experimental work was done without the knowledge of the case/control status to avoid any bias. Fifteen percent of randomly selected samples were repeated for each locus genotyping for replication and validity.

Chemicals were supplied by Sigma-Aldrich (St Louis, MO, USA) unless indicated. Synthetic DNA was produced by GeneArt (ThermoFisher, Waltham, MA, USA). DNA manipulation was performed using standard techniques (Sambrook and Russell, 2001 ). PCR was performed using Q5 polymerase (New England Biolabs, Ipswich, MA, USA) with oligonucleotides purchased from Eurofins (Ebersberg, DE). Plasmid DNA was prepared from E. coli by alkaline lysis using a GeneJET Plasmid Miniprep Kit (ThermoFisher, Waltham, MA, USA). Restriction digests were performed using High-Fidelity restriction endonucleases (New England Biolabs, Ipswich, MA, USA). All cloning was verified by Sanger sequencing (Source Biosciences, Nottingham, UK).
E. coli was chemically transformed using High Efficiency Transformation (New England Biolabs, Ipswich, MA, USA). Chemically competent A. tumefaciens was prepared and transformed by the protocol of Holsters et al. (1978) (link).