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Total RNA was extracted from tissues. The Iso-Seq library was prepared according to the Isoform Sequencing protocol (Iso-Seq) using the Clontech SMARTer PCR cDNA Synthesis Kit (Clontech: 634925 or 634926) and the BluePippin Size Selection System protocol as described by Pacific Biosciences with the following modifications. All of our Iso-Seq libraries were prepared from polyA+ RNA purified from total RNA from organisms. For cDNA conversion, 3 ug of total RNA was used as input into the ClontechSMARTer reaction using SMARTer PCR cDNA Synthesis kit (Clontech: 634925 or 634926). Construction of the cDNA library was performed using Clontech SMARTer PCR cDNA Synthesis Kit (Clontech 634925 or 634926) and PrimeSTAR GXL DNA Polymerase (Clontech R050A or R050B). Fragmentation screening of the samples was performed using the BluePippin Size Selection System (Sage Science, Inc.) as above, and the sample library was constructed using the SMRTbellTM Template prep kit 1.0-SPv3 (Pacific Biosciences, lnc.). After the library was constructed, Sequel Binding Kit 2.0, Sequel Sequencing Kit 2.0, and SequelTM SMRT Cell1Mv4Tray supported by Pacific Biosciences were used for sequencing. |
ISO-Seq |
TRANSCRIPTOMIC |
other |
SINGLE
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