GSM1849363: Hind wing anti Ubx ChIP pulldown rep2; Bombyx mori; ChIP-Seq
Fore and Hind wing buds (80 each) were isolated from second and third thoracic segments of the fourth instar larvae after temporarily aneasthetizing them on ice.The buds were isolated in cold PBS with Roche Protease inhibitor cocktail. The Wing buds were subjected to quick hypotonic lysis to obtain nuclei. Chromatin Immunoprecipitation was carried out on equal volumes of wing bud Chromatin for experiment and negative control, while retaining 10% for input normalization control.The experimental pulldown was carried out using anti Bombyx N terminal Ubx antibodies generated by us. The antibodies against the 19 KDa N terminal region of Ubx protein that are specific to Bombyx Ubx were raised in Rabbit and the resultant antiserum obtained was purified against Milipore Prosep Protein A column to isolate the IgG fraction that was used for the anti Ubx ChIP pulldown. The negative control experiment was carried out using the Normal Rabbit IgG antibodies supplied with the Invitrogen Magnify Chromatin Immunoprecipitation kit, this dataset was used to eliminate non specific peaks from the anti Ubx pulldown set. Libraries for ChIP sequencing were constructed according to a modification of the Illumina ChIP Seq library protocol outlined in “Preparing Samples for ChIP Sequencing of DNA” (Part # 11257047, Rev. A, 2007). Briefly, DNA was subjected to a series of enzymatic reactions that repair frayed ends, phosphorylate the fragments, add a single nucleotide A overhang and ligate adaptors (Illumina ChIP Seq Library preparation kit). The libraries were enriched using PCR (18 cycles), post PCR cleanup was performed using Agencourt AMPURE XP beads (Beckman Coulter #A63881). The enriched PCR fragments were size selected using a 2 % Low melting agarose gel and purified using MinElute Gel Extraction Kit (QIAGEN). The prepared libraries were quantified using Nanodrop Spectrophotometer and validated for quality by running an aliquot on High Sensitivity Bioanalyzer Chip (Agilent).
GEO
GSM1849363