Genelute dna miniprep kit

Genomic DNA of Hmga2 + /+, Hmga2−/− and doxycycline inducible MEF was extracted using the GenElute DNA Miniprep kit (Sigma-Aldrich) according to the protocol provided by the manufacturer. Equal amount of total DNA was applied for Real-time PCR analysis. For detection of DNA nicks, primers for DNA nick assay were designed containing the consensus GT or CT sites specific for DNA nicking enzymes^{38 (link)} (Supplementary Table 1). Nick primers were used with SYBR® Green on the Step One plus Real-time PCR system (Applied Biosystems) and normalized to the Ct values obtained within the surrounding ~300 bp DNA region amplified with the flanking primers (Supplementary Table 1). The % of nick DNA was represented as the ratio between: (Nick FWD + Flank RWD) / (Flank FWD + Flank RWD). To determine the directional association of the different ncRNAs associated to the nick DNA area close to the TSS on each target mRNA, we aligned the sequences of the associated ncRNAs using Global Alignment with free end gaps (Geneious 8.1.9, Biomatters Ltd., San Diego, CA). ncRNA:gDNA hybrids were predicted using the RNA hybrid-online server with parameter (MFE < -50 kcal/mol) and consensus alignment was calculated using T-coffee.

Genomic DNA (gDNA) for SNP genotyping and DNA methylation analysis was extracted from cell lysates using the GenElute DNA Miniprep Kit (SigmaAldrich) and DNeasy Blood and Tissue Kit (QIAGEN, Valencia, CA, USA), respectively, according to manufacturer’s protocol. DNA concentrations were determined using the Quant-iT PicoGreen kit (Invitrogen, Burlington, ON, Canada). Total RNA was extracted from cell lysates using the RNeasy Mini Kit (QIAGEN) according to the manufacturer’s protocol, and treated with 6 U DNase I. RNA quality was confirmed to be high for all samples on the Agilent 2100 Bio-Analyzer (Agilent Technologies, Mississauga, ON, Canada), with an RNA integrity number (RIN) range of 8.1 to 10, and concentrations were determined using the Nanodrop ND-1000 (NanoDrop Technologies, Wilmington, DE, USA).