RNA-seq of Gonyleptes sp MCZ139262
We extracted total RNA using TRIzol reagent (Invitrogen). Tissue fragments were disrupted with a drill in 1000 l TRIzol. To facilitate rupture of the tissue, specimens were flash frozen before drilling. After ~10 min of incubation in TRIzol at room temperature (RT), 100 l of BCP (1-bromo-3-chloropropane; Sigma Aldrich) were added to the tube, mixed by vortexing and incubated at RT for ~10 min, before centri- fuging the samples at 20,000 x g and 4C for 15 min. The aqueous upper layer was transferred to a new tube, mixed with 500 l of Isopropanol, incubated overnight at 20 C and centrifuged again at 20,000 x g and 4C for 15 min to precipitate the total RNA. The resulting pellet was washed with 1 ml of 75% ethanol and centri- fuged at 4C and 6000 x g for 5 min. The washes and centrifuge steps were done twice in order to thoroughly clean the RNA pel- let. After the second centrifuge step, the liquid was removed from the tube without disrupting the pellet before letting it air dry for ~10 min. mRNA capture was done with Dynabeads. We assessed the quantity and quality of mRNA with the 2100 Bioanalyzer (Agilent technologies). For cDNA library construction we used the KAPA Stranded mRNA-Seq Kit (Kapa Biosystems Inc.) which in- cludes its own reagents for mRNA capture and library preparation. We measured quantity and quality of input mRNA with the 2100 Bioanalyzer or the 4200 TapeStation (Agilent) and assessed suc- cess of library construction following 815X PCR amplification by measuring DNA concentration with qPCR (Kapa Library Quant Kit) and library fragment size distribution with the 4200 TapeStation. The samples were sequenced as 150-bp paired-end reads on an Illumina HiSeq 2500 platform at the Bauer Core Facility at Harvard University. The sequencing facility demultiplexed and converted raw data from BCL to FASTQ format.