GSM2475107: Oxy_m6dA-IP-seq_replC; Oxytricha trifallax; MeDIP-Seq
Oxytricha DNA N6-methyladenine IP-seq protocol: Native Oxytricha genomic DNA was sheared into 150 bp fragments using a Covaris LE220 ultra-sonicator (Covaris). Samples were gel-purified on a 2% agarose-TAE gel, blunted with Klenow polymerase (New England Biolabs, MA), and purified using MinElute spin columns (QIAGEN). The fragmented DNA was dA-tailed using Klenow Fragment (3' -> 5' exo-) (New England BioLabs) and ligated to Illumina adaptors following manufacturer's instructions. Subsequently, 2.2μg of adaptor-ligated DNA containing m6dA was immunoprecipitated using an anti-N6-methyladenosine antibody (m6A; Synaptic Systems) conjugated to Dynabeads (Life Technologies). The immunoprecipitated and input libraries were respectively treated with proteinase K, phenol:chloroform extracted, and ethanol precipitated. Finally, they were PCR-amplified using Phusion Hot Start polymerase (New England BioLabs) and used for Illumina sequencing. m6dA IP-seq (Single-read sequencing); standard TruSeq Illumina library construction protocol
GEO Accession
GSM2475107