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Total RNA was extracted from tissues. LncRNA was enriched from total RNA by removing rRNA using NEBNext rRNA Depletion Kit (New England Biolabs). Fragmentation buffer was added to break the RNA into fragments approximately 150 nt, and the first strand cDNA was synthesized using hexamer random primers. DNTPs (dUTP was used instead of dTTP to form the second strand containing U bases) and DNA polymerase I was used to form second strand cDNA. AMPure XP beads (Beckman Coulter Genomics, Inc) was used to purify double-stranded cDNA. The purified double-stranded cDNA was end-repaired, A-tailed and ligated to the sequencing adapter. Then the second strand of U-containing cDNA were digested with the USER enzyme so that the final sequence came from the first-strand cDNA, and thereby preserved the strand directionality of the RNA. Finally, PCR amplification was performed and PCR products were purified using AMPure XP beads. After the library is constructed, we used Qubit 3.0 (Thermo Fisher Scientific, Inc.) to perform preliminary quantification, diluted the library, and then used Qsep118 (Precision Biosystems, Inc.) to detect the insert size of the library. If the insert met expectations, then Q-PCR was used to accurately quantify the effective concentration of the library. Qualified libraries were pooled according to the requirements of the effective concentration and target data volume. Clonal clusters were generated with cBot (Illumina Inc., San Diego, CA) and sequenced with Illumina HiSeq XTen (Illumina Inc., San Diego, CA). |
RNA-Seq |
TRANSCRIPTOMIC |
cDNA |
PAIRED
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