title: GSM3592784: H3K9me3_GH_ChIPseq_rep2 H3K9me3_Th_Gp_ChIP_B2_1; Drosophila melanogaster; ChIP-Seq
library_construction_protocol: For each ChIP-seq reaction ~10,000-15,000 mini-INTACT isolated bead-bound nuclei were processed using Low Cell # ChIP kit (Diagenode: C01010070) as per manufacturer's instructions. In brief, nuclei were fixed in 1% formaldehyde for 2 minutes, immediately quenched with Glycine and then lysed using nuclear lysis buffer with protease inhibitor cocktail at room temperature for 5 minutes. PBS was added to dilute the lysate-bead mix and loaded in AFA tubes (Covaris Inc.: 520045) for sonication. Ultra-sonicator (Covaris Inc.: E220) was used to sheer chromatin to ~200 bp length and chromatin was recovered from the supernatant after magnetic separation. We isolated cell bodies of dopaminergic neurons using Fluorescence Activated Cell Sorting (FACS) during the flies' morning activity peak. In brief, brains were dissected from socially-isolated or group-housed flies expressing membrane-tagged GFP and nuclear tdTomato in their dopaminergic neurons. The flies were obtained by crossing flies carrying TH-GAL4 with a stock carrying pJFRC105-10XUAS-IVS-nlstdTomato in VK40 (gift of Barret D. Pfeiffer,  Rubin Lab, Janelia Research Campus) and pJFRC29-10XUAS-IVS-myr::GFP-p10 in AttP40. To account for possible manual bias, dissectors switched their handling of group- or single-housed flies in each session. Dissected brains were digested using Liberase DH (Roche: 5401054001), manually triturated using glass pipettes, and filtered using a Falcon 35 μm cell strainer (Corning: 352235) before sorting. Approximately 1500 dopaminergic neurons were obtained from approximately 30 brains using a BD FACSAria II sorter (BD Biosciences, USA). Total RNA was extracted using the Arctus, PicoPure RNA Isolation Kit (Thermo Fisher Scientific: 12204-01), ERCC spike-in controls were added and cDNA libraries from this material were prepared. ChIP-seq libraries for sequencing were prepared using MicroPlex Library Preparation kit (Diagenode: C05010012) as per manufacturer's instruction. Ovation RNA-seq System V2 (Nugen: 7102) as per manufacturer's instructions.
attributes.tag: GEO Accession
attributes.value: GSM3592784