Ovation mouse rna seq system 1 16

The Ovation® Mouse RNA-Seq System 1-16 (NuGEN Technologies, #0348) was used per manufacturer’s instructions to construct stranded RNA-seq libraries. 100 ng of total RNA was used as input. First and second strands of cDNA were synthesized from total RNA (100 ng) spiked with 1 µl of 1:500 diluted ERCC ExFold RNA Spike-In Mix 2 (Life Technologies, Carlsbad, CA) at the appropriate ratio. Following primer annealing and cDNA synthesis, the products were sheared using Covaris S220 Focused-ultrasonicator (Covaris Inc., Woburn, MA). 130 µl of each sample was sheared according to manufacturer’s instructions. The parameters were set as follows: 10% duty factor, peak power 175 and 200 cycles per burst at 4 °C for 200 seconds to obtain fragment sizes between 150–200 bp. This was followed by end-repair, adaptor index ligation and strand selection. Strand selection was performed by using custom InDA-C primer mixture SS5 Version5 for mice with cytoplasmic and mitochondrial ribosomal RNA depletion. Finally, libraries were amplified using 17 cycles (Mastercycler^® pro, Eppendorf, Hamburg, Germany), and purified with RNAClean XP Agencourt beads.

The Ovation^® Mouse RNA-Seq System 1–16 (NuGEN Technologies, San Carlos, CA) was used to construct RNA-seq libraries according to the manufacturer's instructions. Briefly, 100 ng of total RNA spiked with 1 µl of 1:500 diluted ERCC Mix 1 (Life Technologies, Carlsbad, CA) was used to start cDNA synthesis. Following the primer annealing and cDNA synthesis, the products (130 µl/each sample) were sheared using Covaris S220 Focused-ultrasonicator (Covaris Inc., Woburn, MA). The parameters were set as follows: 10% duty factor, peak power 175 and 200 cycles per burst at 4 °C for 200 seconds to obtain fragment sizes between 150–200 bp. The products were then subject to end-repair, adaptor index ligation and strand selection. A custom InDA-C primer mixture SS5 Version 5 for mouse was used to allow strand selection. Finally, the libraries were amplified on a PCR thermocycler for 17 cycles (Mastercycler^® pro, Eppendorf, Hamburg, Germany), and purified with RNAClean XP Agencourt beads (Beckman Coulter, Indianapolis, IN); and quantified using Qubit dsDNA HS Kit on Qubit 3.0 Fluorometer (Life Technologies, Carlsbad, CA). The quality and peak size were determined using the D1000 ScreenTape on Agilent 2200 TapeStation (Agilent Technologies, Santa Clara, CA).