Tools-Used.md

Tools used in this manuscript

RNA-seq:

• HISAT2¹ to align reads from RNA-seq experiments
• Rsubread² to count transcripts
• DESeq2² for detection of differentially expressed genes
• Morpheus to visualize count data as an interactive heatmap online. Good for quickly visualizing counts for genes of interest (webtool!)
• pheatmap and ComplexHeatmap² to generate heatmaps of the count matrices
• ggVolcanoR to create Volcano Plots of RNA-seq data (webtool!)
• ShinyGO for gene-set enrichment analysis of differentially expressed genes from RNA-seq and generation of lollipop plots (webtool!)
  • An alternative tool is DAVID
• BioTapestry to build and visualize the gene regulatory network models

ChIP-seq:

• ENCODE Transcription Factor and Histone ChIP-Seq processing pipeline¹ to process ChIP-seq replicates and identify highly reproducible peaks.
  • See this document for more details on the pipeline and this website.
  • Here is a great writeup on how the Irreproducibility Discovery Rate (IDR) analysis is used to combine replicates and identify highly reproducible peaks.
• Hierarchical Alignment (HAL) (LiftOver)¹ to map ChIP-seq peak coordinates from reptile genomes to the mouse genome using a multi-species alignment generated by Brant Faircloth.
• GREAT (Genomic Regions Enrichment of Annotations Tool) to assign putative target genes with each mouse peak (webtool!)
  • An alternative tool is Cistrome-GO
• HOMER (Hypergeometric Optimization of Motif EnRichment)¹ to perform motif analyses (findMotifsGenome.pl)
• deepTools³ to generate enrichment heatmaps
• pyGenomeTracks³ to visualize genome browser tracks of peaks of interest
• bedtools¹ to find peak coordinate intersections
• Canva to generate doughnut charts (webtool!) - Final edits made in Illustrator

For ChIP-seq analyses in which only one replicate was generated:

• bwa¹ to align reads
• MACS2¹ to call peaks

Notes:

• ¹ ran on the Sapelo2 cluster of the Georgia Advanced Computing Resource Center (GACRC)
• ² ran in R/ Rstudio
• ³ ran locally at the command line within Linux Mint running in a Virtual Machine

After generation of figures, vector graphics were edited in Adobe Illustrator and assembled for publication in Adobe InDesign. Other photo edits were made in either Adobe Photoshop or Lightroom. (Note: you can open PDF files in Illustrator to edit as vectors).