Tbr2-dependent transcriptomic analysis

Tbr2 is expressed in all ipRGC cells from development to adulthood, and the Tbr2-Opn4 transcriptional cascade is the main pathway regulating ipRGC formation. In this study, we used bulk RNA-seq to compare transcriptome profiles of E15.5 WT and Six3-Cre:Tbr2-f/f retinas, aiming to identify new regulators in ipRGC subtype development. Twelve retinas from E15.5 wildtype and Tbr2f/f;Six3-Cre embryos from multiple litters were pooled and RNA were extracted using RNAeasy mini kit (QIAGEN). Bulk RNA sequencing were performed in Novogene. Sequencing library were prepared using standard Illumina protocol. RNA sequencing were performed using Illumina HiSeq 6000. Illumina Casava was used for basecalling. The RNA-seq reads were mapped to the mouse genome (mm10) via STAR (v2.5). HTSeq (v0.6.1) was used to count the read numbers mapped to each gene. Differential expression analysis of two groups was performed using the DESeq2 R package (v2_1.6.3). The resulting P-values were adjusted using the Benjamini and Hochbergs approach for controlling the false discovery rate. Genes with an adjusted P-value<0.05 were considered as differentially expressed.