RNA-Seq of Takifugu rubripes:testis
After total RNA was extracted from the sample, ribosomal RNA was removed to retain all codingRNA and ncRNA to the maximum extent. The obtained RNA was randomly interrupted into short segments, and then the first strand of cDNA was synthesized with six-base random primers (random hexamers) using the fragmented RNA as template. Then, buffer, dNTPs(dUTP instead of dTTP), RNase H and DNA polymerase I were added to synthesize the second strand of cDNA, which was purified by QiaQuick PCR kit, eluted by EB buffer, repaired at the end, added with base A, added with sequencing linker, and then degraded by UNG(Uracil-N-Glycosylase) enzyme. The fragment size was selected by agarose gel electrophoresis and amplified by PCR. The final sequencing library was sequenced by Illumina HiSeqTM 4000.After total RNA was extracted from the sample, ribosomal RNA was removed to retain all codingRNA and ncRNA to the maximum extent. The obtained RNA was randomly interrupted into short segments, and then the first strand of cDNA was synthesized with six-base random primers (random hexamers) using the fragmented RNA as template. Then, buffer, dNTPs(dUTP instead of dTTP), RNase H and DNA polymerase I were added to synthesize the second strand of cDNA, which was purified by QiaQuick PCR kit, eluted by EB buffer, repaired at the end, added with base A, added with sequencing linker, and then degraded by UNG(Uracil-N-Glycosylase) enzyme. The fragment size was selected by agarose gel electrophoresis and amplified by PCR. The final sequencing library was sequenced by Illumina HiSeqTM 4000.