RNA-seq transcriptome analysis of male and female zebra finch cell lines

Abstract

The derivation of stably cultured cell lines has been critical to the advance of molecular biology. We profiled gene expression in the first two generally available cell lines derived from the zebra finch. Using Illumina RNA-seq, we generated ~93 million reads and mapped the majority to the recently assembled zebra finch genome. Expression of most Ensembl-annotated genes was detected, but over half of the mapped reads aligned outside annotated genes. The male-derived G266 line expressed Z-linked genes at a higher level than did the female-derived ZFTMA line, indicating persistence in culture of the distinctive lack of avian sex chromosome dosage compensation. Although these cell lines were not derived from neural tissue, many neurobiologically relevant genes were expressed, although typically at lower levels than in a reference sample from auditory forebrain. These cell lines recapitulate fundamental songbird biology and will be useful for future studies of songbird gene regulation and function.

Figure 1

Distribution of uniquely mapping RNA-seq reads among genomic compartments. Flanking regions are defined as being within 1kb up or downstream of the Ensembl cDNA. Reads outside of annotated gene models are termed “intergenic” although it is possible likely of these are actually novel genes. The “other” category includes reads of ambiguous mapping location (reads spanning two compartments) as well as reads mapping to annotated telomeres.

Figure 2

Distribution of normalized read count across 13,333 genes that were represented by an average of one read across both cell lines.

Figure 3

Scatterplot of normalized expression level versus log2 fold change generated by DE-Seq analysis. Points colored in red are those show a significant difference in expression at FDR < 0.01. 98 genes are differentially expressed between cell lines and 2120 are differentially expression between the two cell lines and the auditory forebrain sample.

Figure 4

Distribution of fold change estimates for Z-linked (Z) versus autosomal (A) genes. The distribution of fold changes for autosomal genes is centered around zero whereas the distribution of fold changes for Z-linked genes is shifted to right. Z linked genes tend to be more highly expressed in the male G266 cell line than the female ZFTMA cell line.

Figure 5

Heatmap showing clustering by Euclidean distances between two cell line libraries and three auditory lobule (AL) libraries. The two tumor cell lines, although derived from birds of different sexes and tissue from different parts of the birds, were more similar to each other than they were to profiles of the auditory lobule.