Instrument: Illumina HiSeq 2500
Strategy: OTHER
Source: GENOMIC
Selection: other
Layout: SINGLE
Construction protocol: DamIP-seq was performed as described (Xiao et al., 2010, 2012) with modifications. In brief, 10 µg of gDNA were sheared to 100-300bp with a Covaris S220 Focused-Ultrasonicator in 6x16mm µTUBE (Covaris, Cat. 520045). DNA was boiled 10’ at 98°C, cooled 10’ in ice, diluted with 1 volume of ice-cold 2X DamIP buffer (20 mM Tris-Cl pH 7.4, 300 mM NaCl and 0.2% NP40) and incubated O/N at 4°C under rotation with 4 µg of Anti-N6-methyladenosine antibody (Millipore, 1 µg/µl, ABE572). Antibody-DNA complexes were pulled-down with 25 µl of Dynabeads protein-A (Life technologies, 10002D) by 1h incubation at room temperature. Beads were washed four times in 1X DamIP buffer and resuspended in 300 µl of Elution buffer (50 mM Tris-Cl pH 8, 10 mM EDTA and 0.5% SDS) with 40 µg of Proteinase K. After 3h incubation at 48°C, immunoprecipitated single stranded DNA (ssDNA) was extracted with phenol chloroform, ethanol precipitated and resuspended in 17 µl of EB buffer. Libraries were prepared starting from single strand cDNA with the TruSeq RNA sample prep v2 kit (Illumina, RS-122-2001) according to manufacturer’s instructions, but using Nucleospin columns and NTC buffer when required instead of AmpureXP beads.