RNA-seq of adult P. waltl hindlimbs
Indexed Illumina TruSeq Stranded total RNA Zero-Gold libraries (mean insert size 150 bp) from P. waltl body parts and regeneration stages were combined into four pools and sequenced on 4 lanes (one pool per lane) on Illumina HiSeq High Output mode v4, PE 2x125bp at the Science for Life Laboratory, Stockholm. In all cases, RNA was isolated using Trizol after grinding the sample with a mortar and pestle in liquid nitrogen and precipitated overnight in isopropanol. RNA integrity was assessed using Agilent Bioanalyzer. Samples with RIN ≥ 8.0 were used for library preparation. Illumina Paired End (IPE) reads, from genomic DNA and RNA libraries, were quality filtered using the nesoni clip suit (https://github.com/Victorian-Bioinformatics-Consortium/nesoni.git). Sequencing adaptors, read bases with a per-base Phred quality value below 20 and reads shorter than 64 nucleotides were excluded. In the case that one of the two paired ends passed the quality filtered but not the counterpart, the read was kept and designated as an Illumina single end (ISE). Filtered IPE reads were merged with FLASH and these merged pairs were treated as single end reads (ISE).