GSM3387296: H4T6rep2; Schizosaccharomyces pombe; ChIP-Seq
Lysates were prepared using standard bead beating procedure, sonicated to achieve fragmements of 200-400bp andclarified by centrifugation at 17000g. ChIP-Nexus libraries were prepared essentially as described in He et al., 2015, Briefly, protein G-dynabeads bound DNA-protein-complexes were affinity selected using specific antibodies . DNA was end repaired using T4 DNA polymerase, DNA polymerase I large fragment and T4 polynucleotide kinase. A single 3'-A overhang was added using Klenow 3'-5' exo- polymerase. Adapters were ligated and blunted again by Klenow 3'-5' exo- polymerase to fill in the 5' overhang first and then by T4 DNA polymerase to trim possible 3' overhangs. Blunted DNA was then sequentially digested by lambda exonuclease and RecJf. Digested single strand DNA was then eluted, reverse cross-linked and phenol-chloroform purified. Fragments were  then self-circularized by Circligase. An oligonucleotide was hybridized to circularized single DNA for subsequent BamHI digestion in order to linearize the DNA. This linearized single strand DNA was then PCR-amplified using adapter sequences and library was purified and size selected using Ampure XP beads. The sequencing libraries were sequenced following Illumina's work flow.
GEO Accession
GSM3387296