Ercc exfold rna spike in mix 2

The Ovation® Rat RNA-Seq System (NuGEN Technologies, San Carlos, CA) was used per manufacturer’s instructions to construct 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 a final concentration of 1%. Following primer annealing and cDNA synthesis, end-repair, adaptor index ligation, and strand selection was conducted. Barcodes with unique indices was used per sample for multiplexing. Ribosomal RNA depletion was performed by using custom InDA-C primer mixture SS5 V8 for rat. Finally, libraries were amplified for 13 cycles (Mastercycler® pro, Eppendorf, Hamburg, Germany), and purified with Agencourt XP beads (Beckman Coulter, Indianapolis, IN).

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^® Rat RNA-Seq System 1–16 (NuGEN Technologies, San Carlos, CA) was used per manufacturer’s instructions to construct 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 a final concentration of 1%. Following primer annealing and cDNA synthesis, end-repair, adaptor index ligation, and strand selection was conducted. Barcodes with unique indices was used per sample for multiplexing. Ribosomal RNA depletion was performed by using custom InDA-C primer mixture SS5 V8 for rat. Finally, libraries were amplified for 13 cycles (Mastercycler^® pro, Eppendorf, Hamburg, Germany), and purified with Agencourt XP beads (Beckman Coulter, Indianapolis, IN).