Qiaamp dna mini spin kit

Buffy coats were lysed with proteinase K for 2 h and the DNA was extracted using the Qiagen kit QIAamp® DNA Mini spin kit (Ref 56304) and eluted in 120 µL of elution buffer. Gel electrophoresis was used to confirm successful DNA extraction. Genomic DNA extracted from buffy coats was plated into 96-well plates at a concentration of 50 ng/µL (15 µL per well) and sent to Erasmus Medical Centre, Netherlands, for sodium bisulphite conversion and genome-wide methylation analysis using the Illumina Infinium Methylation EPIC Array, which measures DNA methylation across 850,000 CpG sites (referred to as EPIC ‘probes’), spanning promoter regions, gene bodies, and ENCODE-assigned distal regulatory elements. The output of the EPIC array is beta (β) values for each probe, which range from 0 to 1.

Monocytes stored in ATL buffer were thawed and lysed with proteinase K for 2 h, and DNA was extracted using the QIAamp^® DNA Mini spin kit (QIAGEN, Victoria, Australia, 56304) and eluted in 50 µL of elution buffer. Gel electrophoresis was used to confirm successful DNA extraction. Genomic DNA extracted from monocytes was plated into 96-well plates at a concentration of 50 ng/µL with a volume of 15 µL per well, and sent to Erasmus Medical Centre, Netherlands, for sodium bisulfite conversion and genome-wide methylation analysis using the Illumina Infinium Methylation EPIC Array, which measures DNA methylation across 850,000 CpG sites (referred to as EPIC ‘probes’); these span promoter regions, gene bodies, and ENCODE-assigned distal regulatory elements. Briefly, bisulfite conversion involves the treatment of denatured DNA with sodium bisulfite, which deaminates unmethylated cytosines (converting them to uracils), whilst methylated cytosines remain unchanged. An output of this array used in downstream analysis is the beta (β) value, which is an estimation of methylation computed by the ratio of intensities between methylated and unmethylated alleles at the respective locus.

DNA was isolated from filter paper or whole blood using the QIAamp DNA mini-spin kit (QIAGEN, Valencia, CA, USA) or Chelex-100 [19 (link)]. Plasmodium falciparum infection status was further confirmed by two different molecular methods: nested PCR using primers for 18S ribosomal RNA [20 (link)], and photo-induced electron transfer (PET) real-time PCR using specific fluorogenic primers for Plasmodium (genus) and falciparum (species) [21 (link)].