NextSeq 500 paired end sequencing; Single-nucleotide resolution mapping of DNA abasic sites by snAP-seq
Single-nucleotide resolution mapping of DNA abasic sites by snAP-seq
HeLa cells were washed in cold PBS and harvested by scraping into cold PBS followed by centrifugation at 300 x g. DNA from up to 10^6 cells was extracted using a Quick-DNA™ kit (Zymo Research) according to the manufacturer's instructions. Genomic lysis buffer was supplemented with TEMPO (20 mM) to minimise the introduction of oxidative damage and DNA was eluted in Tris-HCl buffer (pH 7.4, 10 mM). Detailed protocol is available in methods section. Briefly, purified DNA was sonicated using a Covaris M220 system, to an average length of 450 bp. Sonicated DNA was treated with HIPS probe 1 (10 mM, 2 hr, RT) and biotinylated using the CuAAC reaction and biotin-PEG3-azide (2 hr, 37 °C). A custom P7 adapter was introduced onto DNA by ligation using a NEBNext Ultra II kit. Labelled DNA was isolated using magnetic streptavidin beads and unbound DNA was removed by washing. The enriched DNA was denatured on the beads using NaOH solution (RT) and then eluted using NaOH solution (70 °C). Eluted DNA was purified once again on magnetic streptavidin beads to remove any non-specific elution. A single primer extension was performed on eluted ssDNA, then a custom P5 adapter was introduced by ligation. Libraries were amplified by PCR.
Experimental Factor: RNA interference
scrambled siRNA
Experimental Factor: stimulus
none