Transcriptional profiling of the peri-pulmonary vein region of the adult mouse left atrium and corresponding Tbx5-dependence

The left atrium consists of three major parts: the peri-pulmonary vein portion, the appendage, and the vestibule. Previous transcriptional profiling of the adult left atrium and identification of the Tbx5-dependent transcriptome has focused on the atrial appendage (Nadadur et al 2016, Science Translational Medicine). In that study, Tbx5 was shown to regulate a gene regulatory network of atrial identity in the appendage and in its absence results in atrial fibrillation. In order to investigate the regional differences in the transcriptome of the left atrium, the left atrial appendage and the peri-pulmonary vein portion from adult mice were compared by RNA-sequencing. Additionally, as Tbx5 is major regulator of atrial identity, the peri-pulmonary vein portion of the atria was likewise examined following removal of Tbx5 using an adult specific conditional knockout of Tbx5. Overall design: Mice harboring the ROSA26 tamoxifen-inducible cre recombinase transgene, Gt(ROSA)26Sortm1(cre/ERT2)Tyj (R26creERT2), were bred with mice harboring the Tbx5tm1Jse allele (Tbx5flox) to generate Tbx5 conditional mutants, R26creERT2/creERT2; Tbx5flox/flox, and controls, R26creERT2/creERT2; Tbx5+/+. Tamoxifen was administered by intraperitoneal injection on three consecutive days in mice 6 weeks of age and tissue samples were harvested 1 week after treatment as previously reported (Nadadur et al 2016 Science Translational Medicine). Transcriptional profiling was performed on the peri-pulmonary vein region of the left atrium. Total RNA was extracted from four controls and 6 conditional mutants. Following rRNA removal, libraries were prepared using the Illumina TruSeq RNA Sample prep kit v2. Samples were pooled in equimolar ratios and sequenced on the Illumina HiSeq4000 platform. Library preparation and sequencing was performed by the Genomics Core Facility at the University of Chicago. The peri-pulmonary vein samples were compared with the previously published corresponding left atrial appendage samples (Nadadur et al 2016 Science Translational Medicine). Both the peri-pulmonary vein and left atrial appendage samples were analyzed in parallel. Reads were aligned to the GRCm38/mm10 build of the Mus musculus genome (retrieved from UCSC May 23, 2012) using TopHat2. Reads were filtered to remove poorly aligned, duplicated, and unmapped reads using BamTools. Post-alignment, post-filtered reads were assigned to transcripts using StringTie. Downstream analysis performed using R. Differential expression testing was performed using edgeR and limma.