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Total RNA was extracted using TRIzol (Invitrogen, USA) following the manufacturer’s instructions, and RNase-free DNase I (Promega, Madison, WI, USA) was used to purify the RNA. The quality and quantity of the RNA were monitored on 1% agarose gels and assessed using a NanoPhotometer® spectrophotometer (Implen, CA, USA), Qubit® RNA Assay Kit in Qubit® 2.0 Fluorometer (Life Technologies, CA, USA), and the RNA Nano 6000.According to the manufacturer’s recommendations, sequencing libraries were generated using the rRNA-depleted RNA by NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina® (NEB, USA). Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, CA, USA). Before sequencing, ribosomal RNA was removed using the Epicentre Ribo-zero™ rRNA Removal Kit (Epicentre, USA), and the rRNA free residues were cleaned by ethanol precipitation.Sequencing was then performed on an Illumina HiSeq 2500, obtaining 150-bp paired-end reads. |
RNA-Seq |
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cDNA |
SINGLE
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