Laboratory workshop RNAseq of tame and aggressive foxes

Laboratory workshop RNA-seq of tame and aggressive foxes Dr A. V. Kukekova Project was supported by FTP grant № 8474 ICG SB RAS 26. 06. 2013

RNA-seq profiling • Brains of 12 tame and 12 aggressive foxes were dissected in Novosibirsk, flash frozen (Prof. L. N. Trut). • Got prefrontal cortex and amygdala from each. • High-throughput RNA sequencing of these 48 samples. • Per sample coverage of 30 million reads. • Edge. R for normalization and statistical testing.

1. 8 Billion RNA-seq reads from 24 individuals x 2 tissues • Remove adapters • Quality filter (>Q 20) • Remove Poly A/T • Remove mt. DNA, r. RNA • Convert to fasta ~1. 4 Billion RNA-seq reads De novo assembly of fox transcriptome Inchworm Run time: 100 hours Chrysalis Run time: 400 hours N 50 > 1182 bp Quantify Graph & Butterfly Run time: 250 hours Grabherr et al. 2011. Nat Biotech. 29: 644 -652.

Annotation of the fox transcriptome (using dog) • blastx of the fox transcripts to known dog transcripts HTR 5 A (Serotonin Receptor 5 A) Spady and Ostrander 2007

Principal Components Analysis: amygdala vs. prefrontal cortex clustering Tame Prefrontal Cortex Aggressive Prefrontal Cortex Tame Amygdala Aggressive Amygdala

Up and down-regulated transcripts Prefrontal cortex Total Number of Transcripts 10% FDR 5% FDR 1% FDR Up-regulated 454 250 125 Down-regulated 871 427 154

Up and down-regulated transcripts Amygdala Total Number of Transcripts 10% FDR 5% FDR 1% FDR Up-regulated 464 203 61 Down-regulated 390 156 52

Differences in gene expression in serotonergic receptors

Changes in genes in the serotonin pathway HTR 5 A (Serotonin Receptor 5 A) log(CPM) • HTR 5 a polymorphisms have been associated with a large number of Tame difference in human and mouse behaviors. Aggressive Amygdala Prefrontal Cortex

Changes in Monoamine Oxidases MAO-B log(CPM) • Monoamine oxidases play an important role in the metabolism of monoamine neurotransmitters, Tame including serotonin and dopamine. Aggressive Amygdala Prefrontal Cortex Synapse

NMDA (glutamate) receptors and long term potentiation ADCY 1 = adenylate cyclase 1 ITPR 3 = inositol 1, 4, 5 -triphosphate receptor, type 3 GRIN 2 A = glutamate receptor, ionotropic, N-methyl D-aspartate 2 A GRIN 2 B = glutamate receptor, ionotropic, N-methyl D-aspartate 2 B CACNA 1 C = voltage dependent Ca channel a 1 c