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<p>Total RNA was isolated from 3 biological replicates of each of the HRDP v5 strains using QIAzol (Qiagen, Valencia, CA, USA).The RNAeasy Plus Universal Midi Kit (Qiagen) was used to separate long (>200 nt) and short (<200 nt, miRNA-enriched) fractions. The long RNA fraction was purified using the RNeasy Mini Kit (Qiagen). Four microliters of a 1:100 dilution of either ERCC Spikeâ€In Mix 1 or Mix 2 (Thermo Fisher Scientific, Wilmington, DE) was added to each extracted RNA sample. Sequencing libraries for the long RNA fraction were constructed using the Illumina TruSeq Stranded Total RNA Sample Preparation Kit with Ribo-Zero ribosomal RNA reduction chemistry (Illumina) in accordance with the manufacturer’s instructions. An Agilent Technologies Bioanalyzer 2100 was utilized to assess sequencing library quality. A loading control was included in each sequencing batch. The loading control was a library generated from an SHR animal. Samples were sequenced (depending on batch either 2X100 or 2x150 paired end reads) on an Ilumina sequencer.</p>
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