Morphine epigenomically regulates behavior through alterations in histone H3 lysine 9 dimethylation in the nucleus accumbens


Table 1. Genome wide basal gene expression in the nucleus accumbens from RNA-seq.

Total RNA was isolated from 5 animals (10 total NAc punches) per replicate with TRIzol according to manufacturer's protocol, and libraries were then prepared using the mRNA sequencing kit from Illumina (San Deigo, CA). Two replicates were sequenced on an Illumina GAII sequencer with a 36 base pairs (bp) read length. The FASTQ files from Illumina's pipeline were analyzed by the TopHat software for quality filtering and alignment to the mm9 reference genome45. Gene expression levels were then determined by calculating the FPKM (Fragments Per Kilobase of exon model per Million mapped fragments) using the software Cufflinks44. The four expression groups were defined by their FPKM scores using the following criteria: low expression = 0; medium low expression is greater than 0 but less or equal to 10; medium high expression is greater than 10 but less or equal to 100; high expression is greater than 100.

Table 2. Genome wide morphine induced H3K9me2 differential sites.

Mouse NAc punches from animals treated with saline and morphine were crossed-linked and immunoprecipiated with H3K9me2 antibody. Resulting immunoprecipitated DNA and total (input) genomic DNA were prepared for ChIP sequencing using an Illumina kit according to the manufacturer's instructions and sequenced on an Illumina HiSeq 2000 with 50 bp read length.
The MACS (Model-based Analysis of ChIP-Seq data) peak finding algorithm was used to identify H3K9me2 peaks in ChIP libraries generated from both saline- and morphine-treated animals. Differential analysis between morphine and saline groups was carried out with an in-house program called diffReps.



nestler lab
Laboratory of Molecular Psychiatry