title: GSM2585292: Ez_PolII-GA0200_ChIPSeq; Drosophila melanogaster; ChIP-Seq
library_construction_protocol: For each modified histone and Pol II ChIP experiment, we dissected 200 pairs of bam testes and 200 pairs of E(z)61 bam testes in cold PBS and grouped them in 200ml PBS which contained protease inhibitor (Roche complete mini, # 11836153001) and 0.5mM PMSF (MP Biomedicals, #195381). We then added 5.5ml 37% fresh formaldehyde (Supelco, # 47083-U) and incubated at 37oC for 15 mins. The testes were washed twice with 450ml cold 1x PBS (with inhibitors and PMSF). Then 200ml lysis buffer (50mM Tris-HCl, pH7.6, 1mM CaCl2, 0.2% Triton X-100, 5mM butyrate, 1x proteinase inhibitor cocktail, and 0.5mM fresh PMSF) was added and the tissues were homogenized thoroughly followed by incubation at room temperature (RT) for 10 mins. Chromatin was sheared into approximately 200 bp fragments by sonication using Microtip (Misonix, Inc, Microson XL-2000). Chromatin was prepared and ChIP-seq experiments were performed similarly as described previously (Barski A, et al. Cell 2007) with the following antibodies: Pol II (Abcam ab5408), H3K4me3 (Abcam ab8580). ChIP-seq library construction experiments were performed similarly as described previously (Barski A, et al. Cell 2007). Briefly, PNK and Klenow enzymes were used for ChIP DNA end repair, and Taq polymerase used for generating a protruding 3′ A base. After Solexa adaptor ligation, the ChIP DNA was amplified using the adaptor primers for 17 cycles, around 220 bp fragments were isolated from agarose gel. The purified DNAs were used directly for cluster generation and sequencing analysis using the Solexa 1G Genome Analyzer following manufacturer protocols.
attributes.tag: GEO Accession
attributes.value: GSM2585292