|Total RNA input must be between 500 pg and 100 ng. Total RNA inputs above
100 ng per reaction may inhibit amplification, while inputs of less than 500 pg will potentially result in insufficient yields depending on the requirements of the
|RNA samples for use with the Ovation RNA-Seq System V2 must be stored at –80°C.
Avoid frequent freeze/thaw cycles, or RNA degradation may result.
Only CM >90% purity determined by cTnT FACS were used as biological replicates. Total RNA was Trizol-extracted (Invitrogen) and further purified using the Qiagen RNAeasy Micro Kit with DnaseI in-column treatment. cDNA was prepared with the NuGen Ovation RNA-seq System V2 Kit. Total RNA (50 ng) was reverse transcribed to synthesize the first-strand cDNA using a combination of random hexamers and a poly-T chimeric primer. The RNA template was then partially degraded by heating, and the second strand cDNA was synthesized using DNA polymerase. The double-stranded DNA was then amplified using single primer isothermal amplification (SPIA). In this linear cDNA amplification process, RNase H degraded RNA in DNA/RNA heteroduplex at the 5′-end of the double-stranded DNA, after which the SPIA primer bound to the cDNA and the polymerase started replication at the 3′-end of the primer by displacement of the existing forward strand. Random hexamers were then used to amplify the second-strand cDNA linearly. Finally, libraries from the SPIA amplified cDNA were made using the NuGen Ovation Ultralow DR Kit. The mRNA-seq libraries were analyzed by Agilent Bioanalyzer and quantified using an Illumina Library Quantification Kit (KAPA Biosystems). Libraries were prepared by the Gladstone Genomics Core (http://labs.gladstone.ucsf.edu/genomics/home). Four mRNA-seq libraries were pooled per lane of paired-end 100 bp sequencing (High output) on an Illumina HiSeq 2500 instrument (http://humangenetics.ucsf.edu/genomics-services/sample-processing/). Full paper
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