title: GSM9054860: DOG-D4-3_S42; Canis lupus familiaris; RNA-Seq
library_construction_protocol: Total RNA was extracted using TRIzol reagent (Invitrogen) and the Direct-zol RNA Miniprep (Zymo, USA) following the manufacturer's protocol with slight modifications detailed here. MPS Scaffolds from each experiment were preserved in RNAlater at -80°C until RNA extraction. On the day of extraction, samples were thawed at room temperature, and each scaffold was cut into two pieces before transferring to a 1.5 mL microcentrifuge tube. Next, 700 µL of TRIzol reagent was added to the samples, which were then incubated at room temperature for 20 minutes. During incubation, the samples were vortexed for 30 seconds every 5 minutes to ensure complete lysis. The scaffolds were removed from the microcentrifuge and the RNA was precipitated using equal volume of 100% molecular grade ethanol, then transferred to a Zymo-Spin™ IICR column. The remainder of the extraction was performed according to the standard protocol. RNA was eluted in 60 uL of DNAase/RNAse free water. mRNA was isolated from 150ng total RNA using a Nextflex Poly-A Selection kit. Unique dual indexed cDNA libraries were prepared on a Sciclone NGSx liquid handler, using a Nextflex Rapid Directional RNA 2.0 kit miniaturized to 2/5 reaction volume. Optimal PCR cycle count was empirically determined to be 17 by cycling a pool of samples to the plateau phase on a real-time PCR machine. The concentration of libraries was determined using a Molecular Devices (San Jose, CA) Spectramax M2 fluorescent plate reader and Thermo Fisher Scientific (Waltham, MA) Picogreen Assay. Libraries were then pooled by equal mass using a Janus Mini liquid handler. The libraries were pooled by equal mass. The pool was sequenced on a Illumina (San Diego, CA) NovaSeq X+ 1.5B flowcell using the 2x150 bp recipe.