Detection of DNA 5-Methylcytosine (5mC) by Nanopore Sequencing Protocol

Genomic DNA Extraction

Phenol–Chloroform Genomic DNA Extraction Followed by Ethanol Precipitation

1. Homogenize zebrafish embryos in 200 μL homogenization buffer and incubate the sample at 55℃ for no more than 2 h. Invert tube several times to ensure embryos are homogenized following incubation.
2. To remove RNA contamination, incubate sample at 95℃ for 10 min. Add 1 μL RNase A per 100 μL homogenized genomic DNA and incubate at room temperature for 30 min.
3. Perform two phenol–chloroform extractions on samples. Add phenol–chloroform–isoamyl alcohol to sample in 1:1 ratio (approximately 200 μL). Invert tube several times to mix solutions. Spin for 5 min at 13,000 rpm (16,000 rcf).
4. Remove approximately 180 μL from the aqueous upper layer of the mixed solutions and transfer to a new Eppendorf tube, being cautious not to remove any solution from the phenol–chloroform–isoamyl alcohol phase. Repeat phenol–chloroform extraction on the separated aqueous layer.
5. Add 1/10 volume 3 M sodium acetate (pH 5.2), 2.5 volumes ice-cold absolute ethanol and 2 μL linear acrylamide to genomic DNA. Precipitate at -80℃ for 30 min to 1 h or at -20℃ for at least 1 h. Extended precipitation periods (such as overnight) may increase recovery.
6. Following precipitation, spin samples at full speed for 5 min. Remove supernatant and wash pellet in 500 μL cold 70% ethanol. Spin samples at full speed for 5 min. Remove supernatant.
7. Resuspend pellet in nuclease-free water or elution buffer to desired volume. Store at -20 ℃.

Quality Control

1. Use a sensitive fluorometric assay according to manufacturer's instructions to accurately quantify the amount of genomic DNA present in your sample. At least 1 μg of gDNA should be present in your sample. Use NanoDrop 2000 Spectrophotometer to determine purity of the gDNA sample. The following parameters should be met when sequencing a sample.
2. Run gDNA on a TapeStation using Genomic DNA ScreenTape Analysis reagents to determine the size distribution of the DNA fragments. If DNA fragments shorter than 1 kb are present, proceed to step 3. If DNA fragments are larger than at least 1 kb, proceed to Library Preparation and Sequencing.
3. DNA fragments shorter than 1 kb can be removed according to manufacturer's instructions. This protocol performs near complete depletion of fragments less than 5 kb and progressive depletion of fragments less than 10 kb. The recovery efficiency is approximately 50–90% of input DNA. To ensure sample meets all requirements for size selection, repeat all quality control steps detailed in Quality Control following size selection.

Library Preparation and Sequencing

Library preparation and sequencing is performed using the Ligation Sequencing Kit according to manufacturer's instructions. Alternatively, library preparation and sequencing can be performed at a facility that offers Nanopore sequencing services.

Reference:

1. Angeloni A, Ferguson J, Bogdanovic O. Nanopore Sequencing and Data Analysis for Base-Resolution Genome-Wide 5-Methylcytosine Profiling[M]//Chromatin. Humana, New York, NY, 2022: 75-94.