Ercc exfold rna spike in mix 1

To construct RNA-sequencing libraries, an Ovation Mouse RNA-Seq System 1–96 (NuGEN Technologies, Redwood City, CA, USA) was used per manufacturer’s instructions. RNA integrity was determined using a 2100 Bioanalyzer (Agilent) with RIN values > 8. 100 ng of total RNA was used as input. cDNA (first and second strands) was synthesized from total RNA spiked with ERCC ExFold RNA Spike-In Mix 1 (Life Technologies, Carlsbad, CA, USA) at the appropriate ratio. Products were sheared using Covaris S220 Focused-ultrasonicator (Covaris Inc., Woburn, MA, USA) to obtain fragment sizes between 150–200 bp. Followed by end-repair, adaptor index ligation and strand selection. For multiplexing, barcodes with unique indices out of 96, were used per sample. Custom InDA-C primer mixture SS5 Version5 for mice (NuGEN Technologies) was used for strand selection. Libraries were amplified by PCR (17 cycles) on a Mastercycler Pro (Eppendorf) and purified with RNAClean XP Agencourt beads (Beckman Coulter, Pasadena, CA, USA). Libraries were sequenced on a HiSeq 4000 (Illumina, Mira Loma, CA, USA) to generate 15–30 M 75-bp single end reads per sample. Raw data are available at NASA GeneLab (genelab.nasa.gov, accession GLDS-211)²³ .

The Ovation Mouse RNA-Seq System 1–96 (NuGEN Technologies, Redwood City, CA, USA) was used per manufacturer’s instructions to construct RNA-Seq libraries. A total of 100 ng of total RNA was used as input. First and second strands of cDNA were synthesized from total RNA spiked with 1 µL of 1:500 diluted ERCC ExFold RNA Spike-In Mix 1 (Life Technologies, Carlsbad, CA, USA) at the appropriate ratio. Following primer annealing and cDNA synthesis, the products were sheared using Covaris S220 Focused-ultrasonicator (Covaris Inc., Woburn, MA, USA) to obtain fragment sizes between 150–200 bp. This was followed by end-repair, adaptor index ligation and strand selection. Barcodes with unique indices out of 96 indices, were used per sample for multiplexing. Strand selection was performed using a custom InDA-C primer mixture SS5 Version5 for mice (NuGEN Technologies). Libraries were amplified by PCR with 17 cycles on a Mastercycler Pro (Eppendorf) and purified with RNAClean XP Agencourt beads (Beckman Coulter, Pasadena, CA, USA). These libraries were sequenced on a HiSeq 4000 (Illumina, Mira Loma, CA, USA) to generate 15–30 M 75-bp single end reads per sample. Raw data are available at NASA GeneLab (genelab.nasa.gov, accession GLDS-202) [14 ].