| Library |
| Library name |
Construction protocol |
Strategy |
Source |
Selection |
Layout |
|
A total amount of 3 μg RNA per sample was used as initial material for the RNA sample preparations. Ribosomal RNA was removed using Epicentre Ribo-Zero™ Gold Kits (Epicentre, USA). Subsequently, the sequencing libraries were generated following manufacturer recommendations with varied index label by NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina (NEB, Ispawich, USA) . The details of library construction showed as follow: Firstly, ribosomal RNA was removed by kits, RNA fragmentation and short RNA strands were carried out by NEB Next First Strand Synthesis Reaction Buffer under elevated temperature. Subsequently，First cDNA strand was synthesized using random hexamer primers and RNA fragments as template. Second strand cDNA synthesis was subsequently performed using buffer, dNTPs, DNA polymerase I and RNase H. The library fragments were purified with QiaQuick PCR kits and elution with EB buffer, then terminal repair, add poly(A)and adapter were implemented. In order to select cDNA fragments of preferentially 300bp in length, the library fragments were purified and the UNG enzyme was used to digest second strand of cDNA. PCR was performed, aimed products were to retrieve, and the library was completed. RNA concentration of library was measured using Qubit® RNA Assay Kit in Qubit® 2.0 to preliminary quantify and then dilute to 1ng/μl. Insert size was assessed using the Agilent Bio analyzer 2100 system(Agilent Technologies, CA, USA), and qualified insert size was accurate quantitated using Taqman fluorescence probe of AB Step One Plus Real-Time PCR system (Library valid concentration>10nM). The clustering of the index-coded samples was performed on a cBot cluster generation system using TruSeq PE Cluster Kit v4-cBot-HS (Illumina) according to the manufacturer’s instructions. After cluster generation, the libraries were sequenced on an Illumina platform and 150 bp paired-end reads were generated. |
RNA-Seq |
TRANSCRIPTOMIC |
cDNA |
PAIRED
|
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