title: GSM3931419: W_40.7; Homo sapiens; Bisulfite-Seq
library_construction_protocol: Extraction was performed using roughly 10-20 mg of pooled mitochondrial sample. Extraction of mtDNA was similar to the Clayton and Shadel purification method, performed using the mtDNA isolation kit (Abcam) with modifications to the manufacturers' protocol: dounce homogenization was performed for 25 strokes at 4oC, an additional low speed spin at 700g was performed to improve the removal of cell debris and nuclei, 1 µl of proteinase K (Fermentas) was added to the enzyme mix to degrade DNAase, and a phenol-chloroform extraction was included prior to ethanol precipitation to increase the purity of the final DNA solution. Bisulfite treatment of mtDNA was performed using the EpiTect Bisulfite Kit (QIAgen). 200 ng (PFC samples) or 1 µg (cell line samples) of DNA was first converted using the “Bisulfite DNA conversion” steps of the manufacturers protocol “Sodium Bisulfite Conversion of Unmethylated Cytosines in DNA”, with the optional additional cycle added to the conversion program (a final denaturation step for 5 min at 95oC, followed by 2 h at 60oC, and then hold at 20oC). To ensure the maximum recovery of DNA, cleanup was performed using the “Cleanup of bisulfite converted DNA” steps of the manufacturers' protocol “Sodium Bisulfite Conversion of Unmethylated Cytosines in Small Amounts of Fragmented DNA” and using the optional carrier RNA during spin column cleanup. Contaminating RNA for PFC samples was removed with 1 µl RNase A (QIAgen) for 10 min at 70oC with shaking in a total volume adjusted to 200 µl with ddH2O. RNase was subsequently removed by phenol-chloroform extraction and DNA was isolated by isopropanol precipitation.
attributes.tag: GEO Accession
attributes.value: GSM3931419