Publication protocol
"Selective inhibition of HpaII over MspI restriction cleavage:
Unless otherwise stated, 60 ng of DNA was digested with 10 units of either HpaII or MspI
(New England Biolabs) in 1X CutSmart Buffer at 37°C for 1 hour and heat inactivated at
80°C for 30 minutes. Alternatively, NEBuffer 1 was used.
Selective inhibition of bisulfite-mediated deamination of cytosine to uracil:
Three different commercially available kits were evaluated, since the kits differ in efficiency
(izzi (Izzi, Binder et al. 2014): Zymo Research EZ Methylation kit (Nordic BioSite, Täby,
Sweden); EpiJET Bisulfite Conversion Kit (Waltham, MA) and the EpiTect Bisulphite Kit
(Qiagen) were used for the bisulphite conversions according to the manufacturer’s
instructions. Where specified, linearization of DNA prior to conversion was achieved by
digestion of 500 ng of DNA with 20 units of BmtI HF (New England Biolabs) in 1X
CutSmart Buffer at 37°C for 1 hour followed by heat inactivation at 65°C for 30 minutes.
Linearization was confirmed by qPCR using Bmt-Ctr for and Bmt-Ctr rev primers, flanking
one of the two sites for Bmt1 HF in mtDNA. The bisulphite converted DNA was amplified by
PCR with primers specific to the converted mtDNA light (L) and heavy (H) strands (BS-12-L
for + rev and BS-COX1-H for + rev, respectively). The PCR products were purified, and
sequenced by GATC Biotech Laboratories. Data analysis was carried out using the Chromas
Lite 2.1.1 software from Technelysium (South Brisbane, Australia) 5hmC detection by glucosylation and immunoprecipitation of the resulting 5-glucosyl
methylcytosine (5gmC) - containing DNA:
DNA to be analysed was fragmented by sonication with LABSONIC® M from Sartorius
(Goettingen, Germany) (cycle 0.5, amplitude 30 %) for 30 seconds followed by 30 seconds on
ice repeated in total 3 times, to yield an approximate fragment size of 500 bp, as verified by
qPCR analysis with 12S.494bp primers. Subsequent procedure was performed according to
the manufactorer’s description (The Quest 5-hmC™ DNA Enrichment kit from Zymo
Research) with some modifications. Briefly, the fragmented DNA (1.5 µg) was glucosylated
by T4 Phage β-glucosyltransferase (New England Biolabs) in 5-hmC GT Reaction Buffer at
37°C for 1 hour before the addition of 500 µL of JBP Binding Buffer and 10 µL of JBP
Capture MagBeads (Zymo Research). The reactions were incubated with end-over-end
rotation at room temperature for 2 hours. The beads were then washed three times with JBP
Binding Buffer before elution of 5hmC enriched DNA.
"
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