Mimulus guttatus RIL 94 C1b Transgenerational Wound Response Transcriptome
We isolated total RNA from above ground seedling tissue (30-40 mg per seedling) using Direct-zol RNA Mini-Prep following manufacturer’s instructions (Zymo Research Corporation, Irvine, CA), and DNase-treated total RNA with Turbo DNA-free (Applied Biosystems/Ambion, Austin, TX, USA). DNase-treated RNA was extracted from 12 individuals for RNA-sequencing – two individuals from each of three damage-treated and control maternal lines. We prepared RNA-seq libraries using Illumina TruSeq RNA Sample Preparation Kit following manufacturer’s instructions (Illumina Incorporated, San Diego, CA, USA). We Illumina indexed and sequenced (100 bp paired end reads) the twelve libraries on four hiSeq2500 lanes (six samples per lane, each sample run in two lanes).   Reads from each of our twelve samples were mapped to the M. guttatus 2.0 reference genome (http://www.phytozome.net/mimulus_er.php) using the CLC Large Gap Read Mapping algorithm. We used genome annotations to guide the mapping of reads that spanned introns.  Reads mapped to 24,919 of 27,851 annotated transcripts. Down-stream differential expression analyses were based on quantification of reads mapped to the 24,919 annotated genes.