CAGE-seq

We applied cap analysis gene expression (CAGE) to identify the location and relative expression of TSS regions of the PT across both LP and SP. When combined with IsoSeq and RNASeq derived transcript annotation this provided a comprehensive identification of TSS in the genome which allowed us to more accurately apply DNA binding motif analysis to promoter regions. Libraries were prepared according to Hazuki et al.³² and sequenced on an Illumina HiSeq 2500 using V4 chemistry on a 50 cycle Single end sequencing run. We sequenced archived RNA samples from the PT in both SP and LP (ZT4, week 12)²⁹. We also sequenced RNA from PD (both SP and LP), and Pineal for comparison as outgroups. Reads were trimmed using fastx toolkit 0.0.14 and cutadapt 1.4. Reads were mapped using BWA 0.7.17 to the 5^th release of the sheep genome (Oar_rambouillet_v1.0; assembly GCA_002742125.1). CAGEr 1.26.0 was used for processing and cluster analysis of TSS (Supplementary Data ⁴). We filtered reads for a mapping quality >30 and sequencing quality >20. Tag counts were normalized using the power law method with an alpha of 1.12 and T of 10⁶ (deterimined by plotting the reverse cumulatives of PT samples). We clustering TSSs with >1 tags per million (TPM) together using the distclu methods allowing a max distance between TSS of 20 nucleotides.