(A) Overview of methodology for annotating the Schmidtea mediterranea asexual genome based on expression. One hundred sixty-four RNA-seq data sets, four de novo transcriptome assemblies, NCBI complete CDS sequences, and Smed Unigenes were mapped to the SmedGD Asxl v1.1 genome. Reference assemblies were merged, cleaned to remove potential splice variant redundancies, and the best representative transcript for each genomic locus was chosen. Strand information was obtained by BLAST to UniProt, prediction of longest ORF, and data from strand-specific libraries. This process yielded a total set of 38,771 loci. (B) Methodology for gating X1, X2, Xins cell populations based on nuclear to cytoplasmic ratio during Fluorescent Activated Cell Sorting (FACS). (C) Diagram depicting how X1, X2, Xins FACS population gates relate to cell cycle and differentiation stage. (D) Overview of methodology for categorization of annotated loci based on FACS RNA-seq data sets documented in Supplemental Figure 2. FACS RNA-seq data sets were mapped to our annotated genome using Kallisto and normalized with Sleuth. Normalization was done individually for each of the laboratories’ data sets. Normalized TPMs were converted to proportions between available FACS categories of each laboratory, and a final consensus X1:X2:Xins proportion was calculated. (E) A table presenting number of loci in different FACS classification groups, as well as number of protein-coding genes in each group based on Transdecoder evidence.
