Illumina HiSeq 2500 sequencing; RNA seq from plasma exosome before and after chemoradiotherapy for cervical cancer patients
RNA seq from plasma exosome before and after chemoradiotherapy for cervical cancer patients
Exosome was isolated from human plasma by mixing the plasma with Exo2D RNA solution (Exosomeplus).  Exosomal RNA from plasma-derived exosomes was extracted using the miRNeasy Serum/Plasma Kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. The extracted RNA concentration was calculated by Quant-IT RiboGreen(Invitrogen). RNA  size was confirmed using Agilent RNA 6000 Pico Kit and Small RNA Kit on Agilent 2100 Bioanalyzer (Agilent Technologies, Böblingen, Germany).  The 10ng of RNA isolated from each sample was used to construct sequencing libraries with the SMARTer smRNA-Seq Kit for Illumina, following the manufacturer's protocol. Briefly, Input RNA is first polyadenylated in order to provide a priming sequence for an oligo(dT) primer. cDNA synthesis is primed by the 3' smRNA dT Primer, which incorporates an adapter sequence at the 5'end of each RNA template, it adds non templated nucleotides which are bound by the SMRT smRNA Oligo-enhanced with locked nucleic acid (LNA) technology for greater sensitivity. In the template-switching step, PrimeScript RT uses the SMART smRNA Oligo as a template for the addition of a second adapter sequence to the 3'end of each first-strand cDNA molecule. In the next step, full-length Illumina adapters (including index sequences for sample multiplexing) are added during PCR amplification. The Forward PCR Primer binds to the sequence added by the SMART smRNA Oligo, while the Reverse PCR Primer binds to the sequence added by the 3' smRNA dT Primer.  The amplified libraries were purified from 6% Novex TBE-PAGE gels (Thermo Fisher, MA) to excise over than 138 bp (over than 18 bp of cDNA plus 120 bp of adaptors) fraction Resulting library cDNA molecules include sequences required for clustering on an Illumina flow cell.