title: GSM1430011: Twist ChIP-nexus (Drosophila embryos) Rep 2; Drosophila melanogaster; OTHER
library_construction_protocol: embryos were cross-linked in 1.8% formaldehyde. Embryos were then dounced to break down cells and extract nuclei. Chromatin was sonicated by bioruptor to an average size of 300~500 bp. 300 ul soluble chromatin from ~100 mg embryos was used DNA-protein-complexes were isolated with specific antibodies when still being attached to protein A-dynabeads. DNA was end repaired using T4 DNA polymerase, DNA polymerase I large fragment (Klenow polymerase) and T4 polynucleotide kinase. A single 3'-A overhang was added to the blunted ends using Klenow 3'-5' exo- polymerase. Adapters with single 3'-T overhangs and 5' overhangs (barcode composed of 5 random nucleotides) were ligated to the adenylated fragments. Ligated fragments were blunted again by Klenow 3'-5' exo- polymerase to fill in the 5' overhang first and then by T4 DNA polymerase to trim any possible 3' overhang. Blunted DNA was subsequently digested by lambda exonuclease first and RecJf afterwards. Digested single strand DNA was then eluted, reverse cross-linked and purified prior to self-circularization by Circligase. An oligonucleotide was mixed with circularized single DNA for subsequent BamHI digestion to linearize the single DNA again. Linearized single strand DNA was then PCR-amplified using adaptor sequences and library was purified on 2% agarose gel to remove adaptor-adaptor ligation products. Following cluster generation on the flowcell surface, the sequencing libraries were sequenced following Illumina's protocol.