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RNA integrity was checked with an Agilent 2100 Bioanalyzer Instrument using the RNA 6000 Nano kit and sample quality with RIN > 7 was used for Iso-Seq library preparation. The first-strand cDNA synthesis was performed and the second-strand cDNA synthesis was also subsequently conducted according to PacBio instructions. Following PCR cycle optimization, PCR amplification was performed. Then, PCR products were purified with SMRTbell cleanup beads. The amplified library was subjected to the following steps: end repairing, A-tailing, SMRTbell adapter ligation, Nuclease treatment, and purification. After these processes the final sequencing library was produced. At last, quality accession of the library was implemented using Agilent 2100 Bioanalyzer Instrument and Qubit fluorometer. The final library was sequenced with the PacBio sequencing platform in Novogene Bioinformatics Technology Co., Ltd (Beijing, China). |
ISO-Seq |
TRANSCRIPTOMIC |
cDNA |
SINGLE
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