Nebnext multiplex small rna library prep

TRIzol (Thermo Fisher, Shanghai, China) used to extract total RNA from the tissue samples. RNA quantity and purity were determined using Agilent 2100 Bioanalyzer and the RNA 6000 Nano Labchip Kit (Agilent, Santa Clara, USA). Epicentre Ribo-Zero™rRNA Removal Kit (Epicentre, America) were used to remove rRNA before the RNA-seq library construction. Then, the rRNA-depleted RNAs were then fragmented and reverse-transcribed to obtain cDNA libraries using the TruseqTM RNA sample prep Kit (Illumina, America). Small RNA-seq library construction was performed according to the instructions of NEBNext Multiplex Small RNA library Prep (Illumina, America), which was followed by TRIzol extraction. Then, the library was amplified and enriched using PCR technique, proprietary indexed adapters were then ligated to 5ʹ- and 3ʹ-termini, and later electrophoresed using 15% concentration of agarose gel to obtain the target fragments. Finally, all sequenced libraries were sent to the sequencing company (Personalbio, Shanghai, China) and sequenced by Illumina Hiseq4000 platform for paired-end sequencing after quality control.

Poly-adenylated RNA was isolated with Oliogtex (QIAGEN). rRNA was depleted from total RNA with Ribo-Zero Gold (EpiCentre). Libraries were prepared using the Illumina Directional mRNA-Seq Sample Prep and the NEBNext Multiplex Small RNA Library Prep kits and then sequenced on an Illumina HiSeq 2500. RNA-seq reads were filtered for quality and to remove adapters, aligned to hg19 using TopHat2, and transcripts identified with Cufflinks v2.1.1 using default settings.