title: RNA seq of Patella vulgar: Radula Sac taken from Limpet 3
design: Limpets of the species Patella vulgata were selected from shoreline a site adjacent to the Institute of Marine Sciences, University of Portsmouth, UK. To reduce biological variation, female limpets of a similar size were selected. Shell diameter at the widest point was between 40 and 48mm, and radula length was 75 to 87mm. Ten samples were collected and were dissected to collect matched samples for each of the 6 tissues of interest: foot muscle and the radula which is divided into 5 sub-sections consisting of the radula sac, Stage I in the first 15-20 rows of teeth that were immature and distinguishable by their lack of colour; Stage II in the next 12 rows which were early maturing; Stage III were late maturing in the next 30 rows and the remainder were mature in stage IV. The 5 limpets with the best overall RNA quality across all 6 tissues were selected for sequencing. Tissue was homogenised and snap frozen in QIAzol. Total RNA was extracted from individual samples using the RNeasy Mini Kit from Qiagen according to manufacturers guidelines. RNA quality was assessed by using a ThermoFisher Nanodrop and Agilent Bioanalyzer 2100. RNA integrity ranged from 3.8 to 8 (mean = 6.3 0.9). NGS libraries were created for all 30 samples using the Illumina TruSeq Stranded mRNA library prep kit, and were multiplexed and sequenced by Theragenetex (theragenetex.com) using the Illumina HiSeq 2500 sequencer to produce paired-end 100 bp sequences.