Genes overlapping between current DEGs and all genes differentially expressed between ethanol and control groups from your 2016 study were also under-represented (14 out of 552; < 0.05). Table 1 Functional group over-representation analysis of genes up-regulated in VTA of the decitabine-treated group compared to vehicle control. a cryostat. The ventral tegmental area (VTA) was dissected using the brain-punch tissue set in accordance with coordinates from your mouse brain atlas (Franklin & Paxinos, 2008). Total RNA was isolated from tissue samples using MagMAX?-96 Total RNA Isolation Kit (Thermo Fisher Scientific, Waltham, MA) and sent to the Genomic Sequencing and Analysis Facility (GSAF) at the University or college of Texas (https://wikis.utexas.edu/display/GSAF/Home+Page) for RNA-Seq library preparation. All libraries exceeded the quality control, and samples were sequenced around the Illumina HiSeq 2500 sequencer at ~30 million reads per sample (single-read, 1 50 bp). Quality assessment of data files was carried out using FastQC (v0.11.5). Reads were aligned to the mouse reference (GRCm38/mm10) using TopHat2 (v2.0.10) and mapped using Bowtie2 (v18.104.22.168). HTSeq (v0.6.1) was used to assemble transcripts and generate read counts per transcript using the output from TopHat2. Data were normalized, and genes that were differentially expressed between the Decitabine and Vehicle groups (DEGs) were decided using the DESeq2 package for R. Bioinformatics analysis Two DEG lists, one up- and one down-regulated by decitabine (nominal value < 0.05), were subjected to an over-representation analysis for biological pathways using Enrichr (Chen et al., 2013) (http://amp.pharm.mssm.edu/Enrichr) based on two databases: Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). Over-representation value for each pathway was calculated based on the Fishers exact test with the correction for false discovery rate. To test if DEGs are markers of specific cell types, we profiled the top 41 DEGs (value < 0.0001) using a database for cell type-specific brain transcriptomes (http://web.stanford.edu/group/barres_lab/brain_rnaseq.html) (Zhang, Chen, et al., 2014). At least 3-fold enrichment, compared to a second highest expression, was used as the criterion for a specific cell marker. In addition, to identify specific markers of DA Ligustilide neurons affected by the decitabine treatment, we compared all DEGs to dopamine-enriched genes recognized by our previous study investigating effects of ethanol binge drinking for 3 weeks on microdissected DA neurons (Marballi, Genabai, Blednov, Harris, & Ponomarev, 2016; Appendix S1). We also compared DEGs from the current study to all genes differentially expressed between ethanol and control groups from your 2016 study. Statistical significance for the overlap between gene lists was estimated using a hypergeometric probability test. Electrophysiological experiment In a separate experiment, mice were treated with either decitabine or Ligustilide vehicle (a single injection every other day for 8 days for a total of 4 injections). Twenty-four hours after the last injection, mice were decapitated under isoflurane anesthesia, and brains were rapidly dissected in ice-cold sucrose-substituted artificial cerebrospinal fluid (ACSF). Horizontal midbrain slices 200 m solid were cut on a vibrating microtome and recovered for 1 h at 34 C in oxygenated Ligustilide ACSF (in 126 mM NaCl, 2.5 mM KCl, 1.2 mM NaH2PO4, 1.2 mM or 0.1 mM MgCl2, 2.4 mM CaCl2, 11 mM glucose, 21.4 mM NaHCO3). During recording, slices were perfused with ACSF at 34 C. Recording pipettes were filled with 150 Ligustilide mM NaCl and loose seals (10C20 M ) were created. Putative DA neurons were identified by the presence of slow (1C5 Hz) spontaneous pacemaker-type firing of action potentials of >1.2-ms width in voltage-clamp mode. Firing rates were monitored in current-clamp I = 0 mode. Data were digitized at 10 KHz and filtered at VPREB1 2 KHz. Spikes were detected and analyzed with the AxoGraphX event-detection power. Basal firing rate was recorded from 59 neurons (28 saline, 31 decitabine; n = 8 mice per group). Firing rate response to 80-mM ethanol was recorded.