mouse-raised STAT92E antibody (self-made); 100-150aa polypeptides of STAT92E protein chosen as the antigen
Sequenced DNA Library
About 400 Drosophila adult midguts(with the hindgut and Malpighian excluded) were dissected in ice-cold PBS, and then cross-linked with 1% formaldehyde for 15 minutes. After washing process to remove the formaldehyde, intestinal tissue was lysed with RIPA buffer which contains 1% SDS on ice for 30 minutes. The sonication of chromatin was performed using the Covaris (AFA) system with 3% power output for 5 minutes each on 100μl lysates.STAT92E-bound chromatin fragments were enriched by immunoprecipitation with mouse raised STAT92E antibody. Most chromatin fragments resulting from sonication occurred between 200 and 400 bp.The process of dilution, antibody incubation, protein G pull down, beads washing, DNA complex elution, de-link, RNAse A / protease K digestion and DNA extraction all with normal ChIP protocols.The high throughput sequencing process was carried out using the Illumina solexa system. ChIP and input libraries were prepared using NEXTflex™ Illumina ChIP-Seq Library Prep Kit (Illumina) according to the manufacturer’s protocol. The indexed libraries were pooled together, and sequenced using the Illumina HiSeq 2000 (Illumina) instrument.