Transcriptome library construction and paired-end strand-specific transcriptome (rRNA-free) sequencing

The protocols of transcriptome library construction and paired-end strand-specific transcriptome sequencing were previously reported [79]. Briefly, 3 μg RNA per sample was used for RNA-seq library construction. Firstly, due to some lncRNAs lacking the poly (A) tail, Epicentre Ribo-zero™ rRNA Removal Kit (Epicentre, USA) was used to remove the ribosomal RNA from the total RNA, and ethanol precipitation was used to clean up the rRNA free residue. Subsequently, the rRNA-depleted RNA by NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina® (NEB, USA) was used to produce sequencing libraries according to the manufacturer’s recommendations. Then, the random hexamer primer and M-MuLV Reverse Transcriptase (RNaseH-) were employed to synthesize the first-strand cDNA, and the DNA Polymerase I and RNase H was subsequently used to synthesize the second-strand cDNA. The 3′ ends of DNA fragments was adenylated and NEBNext Adaptor with hairpin loop structure was ligated to prepare for hybridization. With the purpose of selecting cDNA fragments of favorably 250 ~ 300 bp in length, the AMPure XP system (Beckman Coulter, Beverly, USA) was selected to purify the library fragments. Before PCR experiment, 3 μL USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C. In the PCR experiment, the Universal PCR primers, Index (X) Primer and Phusion High-Fidelity DNA polymerase were added in the reaction system. At last, AMPure XP system and the Agilent Bioanalyzer 2100 system was used to purify the products and evaluate the library quality, respectively. A cBot Cluster Generation System using HiSeq 4000 PE Cluster Kit (Illumina, NEB, USA) was used to cluster the index-coded samples following the manufacturer’s instructions. Subsequently, an Illumina Hiseq 4000 platform was used to sequence the library preparations and produced 150 bp paired-end reads.

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