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Total RNA of each sample (n = 3 for brain, eye, inner ear, lung, muscle, and tympanic membrane) was extracted and purified using Trizol (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. After been purified with poly-T oligo-attached magnetic beads, the mRNAs were fragmented. First-strand cDNA was synthesized using random hexamer primers. Second-strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. The remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of the 3’ ends of the DNA fragments, adaptors were ligated to the products. Then, PCR was performed with a HIFI DNA polymerase, universal PCR primers, and Index (X) primer. The library preparations were sequenced on an Illumina Novaseq 6000 platform (PE150 strategy) by Novogene (Beijing). |
RNA-Seq |
TRANSCRIPTOMIC |
RANDOM PCR |
PAIRED
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