Genejet genomic dna gdna purification kit

DNA was extracted from 3-mL cultures of S. meliloti Sm1021 resistant mutants grown overnight using the GeneJET genomic DNA (gDNA) purification kit (Thermo) according to the manufacturer’s protocol. Next-generation sequencing (NGS) libraries were prepared using the NEBNext Ultra II DNA library prep kit (New England BioLabs [NEB]). DNA sequencing was performed on the Illumina MiSeq platform with the 250-bp plus 250-bp paired-end protocol. Library preparation and sequencing were performed at Skoltech Sequencing Core Facilities. Raw reads were filtered and trimmed with Trimmomatic (68 (link)), and genome assembly was performed with SPAdes (69 (link)). The identification of transposon insertion positions was performed by stand-alone BLAST analysis using the Km^r gene sequence as bait (70 (link)). The genome annotation under GenBank accession no. NC_003047 was used as a reference.

Genomic DNA was isolated from thoracic aorta of C57BL/6J mice using GeneJet Genomic DNA (gDNA) purification kit (ThermoFisher Scientific, USA) as per manufacturer’s protocol. To access the genomic methylation pattern, gDNA was deaminated using EpiJet Bisulfite conversion kit (ThermoFisher Scientific, USA) according to manufacturer’s protocol. The deaminated gDNA was further used as template for running methylation specific PCR (MSP Assay). CpG islands were determined in the promoter region of miR34a, several hundred base pairs upstream of precursor transcription start site and 5 sets of primers were used for methylated and unmethylated DNA region each as per [33 (link)]. Real-time PCR was performed with following conditions: 95°C for 10 minutes, followed by 40 cycles of 95°C for 30 seconds, 52°C for 30 seconds, and 60°C for 30 seconds. A reaction tube w/o template was used as negative control and all the samples were run with n = 3 technical replicates.

Genomic DNA was isolated from HUVEC and MDMs using a GeneJet Genomic DNA (gDNA) purification kit (ThermoFisher Scientific, Waltham, MA, USA), as per the manufacturer’s protocol. To access the genomic methylation pattern, gDNA was deaminated using an EpiJet Bisulfite conversion kit (ThermoFisher Scientific, USA), according to the manufacturer’s instructions. The deaminated gDNA was further used as a template for running methylation-specific PCR (MSP Assay). CpG islands were determined in the promoter region of miR-34a, several hundred base pairs upstream of the precursor transcription start site, and 5 sets of primers were used for the methylated and unmethylated DNA regions each [38 (link)]. Real-time PCR was performed with the following conditions: 95 °C for 10 min, followed by 40 cycles of 95 °C for 30 s, 52 °C for 30 s, and 60 °C for 30 s. A reaction tube w/o a template was used as a negative control, and all the samples were run with n = 3 technical replicates.