br Experimental design materials and methods br Acknowledgem
Experimental design, materials and methods
Acknowledgements This research was supported by National Research Foundation of Korea, South Korea (NRF) grants funded by Ministry of Education (2013R1A1A2057932) and Ministry of Science, ICT and Future Planning, South Korea (2016M2B2A4912473).
Data Table 1 showed data on gene expression profile in T47D Amyloid Beta-Peptide (1-40) (genes regulated 1.5 time or higher) transfected with 17β-HSD1 siRNAs (si17B1) or negative control siRNA (NC), and treated with 1nM estradiol (E2), revealed by microarray analysis with contrast NC+E2 vs. si17B1+E2 (see Table 5 for additional information). Table 2 showed the 14 functional terms found in the cancer category of the IPA biological function analysis of 208 genes from three fold change lists (genes which fold change equal or higher than 1.5 in at least one contrast), generated by the three contrasts (NC vs. si17B1; NC vs. NC+E2; and NC+E2 vs. si17B1+E2) of microarray analyses (see Table 5 for contrast description). Table 3 showed the 14 functional terms found in the cell death and survival category of the IPA biological function analysis of 208 genes generated by the three contrasts of microarray analyses. Table 4 showed data on the IPA network analysis of 208 genes from the three contrasts (NC vs. si17B1; NC vs. NC+E2; and NC+E2 vs. si17B1+E2) of the microarray analysis. Figs. 1–4 showed IPA Canonical pathway analyses for interferon signaling pathway in the NC vs. NC+E2 contrast (Fig. 1), antigen presentation pathway in the NC vs. si17B1 contrast (Fig. 2), antigen presentation pathway in the NC vs. NC+E2 contrast (Fig. 3), and role of BRCA1 in DNA damage response in the NC vs. NC+E2 contrast (Fig. 4).
Experimental design, materials and methods
Acknowledgements This work was supported by Canadian Institutes of Health Research, Canada, with grant to S.-X. Lin (Principal Investigator for FRN57892).
Data The dataset of this article comprises three data files as follows: (i) Dataset 1. FASTA file (raw_trinity_assembly.fasta) of the unfiltered Trinity assembly, (ii) Dataset 2. FASTA file (filtered_trinity_cds.fasta) of the filtered Trinity assembly containing 44,857 contigs, and (iii) Table 1: voltage gated like (VGL) ion channel sequences resolved from the Dugesia japonica transcriptome. Contig IDs for ion channel sequences contained in the D. japonica de novo assembly organized by putative VGL ion channel family following manual inspection of transmembrane helix organization, structural motifs and ion selectivity residues. FPKM values reflect expression levels in whole (non-regenerating) animals. Additional analysis of these datasets are presented in the associated publication (‘Utilizing the planarian voltage-gated ion channel transcriptome to resolve a role for a Ca2+ channel in neuromuscular function and regeneration’, Chan et al. ).
Experimental design, materials and methods Sequencing was performed on individuals from a clonal, asexual laboratory strain of the planarian D. japonica (GI strain). In order to sample a diversity of expressed transcripts, total RNA was extracted from intact (non-regenerating) worms (3 biological replicates of 100 individuals), as well as anterior worm fragments harvested at various intervals following tail amputation (1, 12, 24h; 3 biological replicates of 200 heads per time point) using Trizol reagent. mRNA was purified using oligo(dT) beads (Dynal), yielding approximately 2µg mRNA per biological sample. RNA-seq libraries were prepared according to the Illumina mRNA-Seq Sample Prep kit and Illumina TruSeq kit manufacturer protocols. Libraries were sequenced on Illumina HiSeq 2000 machines (Sanger Center, Hinxton) and the resulting 100bp paired end reads were processed with Trimmomatic version 0.22  to remove adapter sequences and low quality reads (sliding window quality filter, window size=4, minimum average quality score=25) while retaining reads ≥50bp. In order to generate the de novo transcriptome assembly, overlapping paired-end reads were merged using FLASH  and fed into the Trinity pipeline , carried out with a minimum k-mer coverage of 2 and default k-mer size of 25. Graphs not resolving within a 6h window were excised to allow the assembly to proceed and the minimum contig or transcript length was set to 100nt. Relative transcript abundance was estimated using bowtie (version 2) to align trimmed reads to the de novo assembly and RSEM (version 1.2.11) to quantify read mapping, yielding FPKM (Fragments Per Kilobase of transcript per Million mapped reads) values for each contig. Assembled contigs were annotated using the TransDecoder package to predict translated open reading frames, which were searched against the NCBI Conserved Domain Database.