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
Leishmania DNA was purchased from ATCC (product 30012D, lot 62762024). Purified and sonicated DNA was treated with UNG (10 U, NEB) for 18 hr at 37 °C. 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
none
Experimental Factor: stimulus
Uracil-DNA Glycosylase; 10 units