GSM3931428: M_25.6_negativeControl_BS; Homo sapiens; Bisulfite-Seq
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. To further assess the accuracy of our mtDNA BS-seq technique, we checked the apparent background of our technique using unmethylated mtDNA prepared from 4 samples (M_25.6, W_27.7, M_46.8, W_50.5) using long range PCR (LATaq, TaKara) to amplify the complete mtDNA genome in two overlapping fragments, A and B (see below). 50 µl PCR reactions were prepared according to the manufacturers' recommendations and reaction conditions were 5 min at 98oC, 40 cycles of denaturation for 30 s at 98oC, annealing for 30 s at 58oC, and extension for 10 min at 72oC, with a final extension for 5 mins at 72oC. Products were agarose gel purified on a 0.7% gel at 120V for 2 hours in 1x TBE and gel extracted (Gel extraction kit, QIAgen). Equimolar amounts of each fragment were mixed, and then BS-seq was performed as for purified mtDNA.  mtfullAF: AACCAAACCCCAAAGACACC  mtfullAR: GCCAATAATGACGTGAAGTCC  mtfullBF: TCCCACTCCTAAACACATCC  mtfullBR: TTTATGGGGTGATGTGAGCC
GEO Accession
GSM3931428