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Total RNA of samples was extracted by the CTAB method. The quality and quantity of each RNA sample were assessed by the NanoDrop 2000 and the Agilent 2100 Bioanalyzer. Only RNA samples with absorption 260/280 ratios from 1.9 to 2.2, 260/230 ratios from 2.0 to 2.5, and RNA integrity number values greater than 6.8 were used for subsequent experiments. The mRNA was enriched by binding of A-T complementary pairing to the plasmidA tail of mRNA after the sample was tested and the magnetic beads with Oligo were used. The fragmentation buffer was added to break the mRNA into short fragments. We used mRNA as a template and then synthesized a single-stranded cDNA using random hexamers. We added buffer, dNTPs and DNA polymerase I to synthesize two-stranded cDNA. The double-stranded cDNA was purified using AMPure XP beads and subjected to end repair, addition of A tail, ligation of the sequencing linker, and fragment size selection. Finally, PCR enrichment was performed to obtain a final cDNA library. |
RNA-Seq |
TRANSCRIPTOMIC |
RT-PCR |
PAIRED
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