title: Illumina MiSeq paired end sequencing; Genome-wide analysis of Groucho in Drosophila BG3 cells
design: Genome-wide analysis of Groucho in Drosophila BG3 cells
library_construction_protocol: Approximately 5-7 x 10^7 cells were fixed at room temperature by incubation with 1% formaldehyde for ten minutes. The fixation reaction was quenched with 200mM Glycine after which cells were washed three times with 1x Phosphate Buffered Saline (1xPBS). This was followed by a ten-minute lysis on ice (Nuclear Lysis Buffer: 50mM Tris-HCL (pH 8.1), 10mM EDTA, 1% SDS, 1xRoche Complete Inhibitor) and the addition of 1.6 volumes of IP Dilution Buffer (20mM Tris-HCL (pH 8.1), 150mM NaCl, 2mM EDTA, 1% TritonX-100, 0.01% SDS, 1xRoche Complete Inhibitor). Chromatin released from lysates was fragmented to a range of 200 to 1000bp using a Diagenode Bioruptor set for 15 pulses (30 seconds ON, 30 seconds OFF). Fragmented chromatin was further diluted (five-fold) in IP dilution buffer and precleared for 3-5 hours with Protein G PLUS-Agarose beads and 3 mg of normal rabbit IgG (Insight Biotechnology and New England Biolabs). Following this precleared chromatin was either snap-frozen and stored at -80C or immediately used for ChIP. For each ChIP experiment an aliquot of chromatin deriving from approximately 1 x 107 cells was used. An aliquot of chromatin was set aside to use as an input control. ChIP samples were treated with the appropriate concentration of an antibody along with Protein G PLUS-Agarose beads and kept on a rocker at 4C overnight. After immunoprecipitation the beads were subjected to a wash with IP Dilution Buffer and two washes each with IP Wash Buffers 1 and 2 (IP Wash Buffer 1: 20mM Tris-HCL (pH 8.1), 50mM NaCl, 2mM EDTA, 1% TritonX-100, 0.1% SDS; IP Wash Buffer 2: 10mM Tris-HCl (pH 8.1), 250mM LiCl, 1mM EDTA, 1% NP-40, 1% deoxycholic acid)). They were finally washed with TE Buffer before bound chromatin-protein complexes were eluted (IP Elution Buffer: 100mM NaHCO3, 1% SDS). Eluted complexes were treated with RNase A (Invitrogen) at 37C for an hour to eliminate RNA, 180mM NaCl at 65C for 4-6 hours to reverse crosslinks, and Proteinase K (Roche) at 45C overnight to remove proteins. The aliquot kept aside as input was subjected to the same treatment.  Purified DNA for ChIP and input was obtained by phenol-chloroform extraction and ethanol precipitation. The concentration of ChIP and Input DNA was checked on the Qubit fluorometer (Invitrogen). Approximately 20-50ng of the purified ChIP and Input DNA was used to generate ChIP libraries using either the NEBnext ChIP-Seq Library Prep Master Mix Kit (E6240S) or the NEBNext Ultra DNA Library Prep Kit (E7370S) according to manufacturers instructions. Adaptor-ligated DNA was amplified with 15 cycles of PCR and fragments of 250-350 bp selected by agarose gel extraction. Resulting libraries were assessed for concentration and size using the Qubit fluorometer and the Agilent2100 Bioanalyzer.