ChIP-Seq with anti H3K9Ac S.mansoni miracidia BRE
DNA (30 ng) from native ChIP was polished to generate blunt ended fragments, add 3’-dA overhangs by treatment with Klenow polymerase and ligated to adapters as described (Pomraning et al., 2008). Fragments were size selected (150 bp to 500 bp) after migration through a 2% NuSieve agarose gel and sequencing libraries were generated by PCR amplification with 1 µl sample DNA, 1 X Phusion polymerase mix (Finnzymes, #F531), 2.25 µM of each Solexa PCR primer. We used a standard protocol: denaturation, 90°C, 30s; amplification (repeated 18 times), 98°C for 10 s, 65°C for 30 s, 72°C for 30 s; final elongation, 72°C, 5min. PCR products were purified on QIAquick PCR Purification columns (Qiagen, #28104) and eluted in 30 µl Qiagen elution buffer. We quantified the DNA library with a Nanodrop1000 spectrophotometer (Thermo Scientific) and clusters were generated with 4 pM of the 10 nM library dilution on the Illumina cluster station. A 36-cycle sequencing kit on the Illumina 1G analyzer was used according to the manufacturer’s instructions.
University of Perpignan
ChIP-Seq_H3K9Ac_Sm_mira_BRE