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After extracting total RNA from the samples, magnetic beads with Oligo(dT) are used to enrich eukaryotic mRNA (for prokaryotes, rRNA is removed using a kit before proceeding to the next step). Fragmentation buffer is then added to break the mRNA into short fragments. Using mRNA as a template, the first strand of cDNA is synthesized with random hexamer primers. Buffer, dNTPs, RNase H, and DNA polymerase I are then added to synthesize the second strand of cDNA. After magnetic bead purification, end repair, poly(A) addition, and sequencing adapter ligation are performed. The fragment size selection is carried out using agarose gel electrophoresis, followed by PCR amplification. The prepared sequencing library is then sequenced using DNBSEQ T7. |
RNA-Seq |
TRANSCRIPTOMIC |
PCR |
PAIRED
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