Homo sapiens
Protocols: 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) fractionResulting library cDNA molecules include sequences required for clustering on an Illumina flow cell.
disease
cervical cancer
individual
AU-19-27
organism
Homo sapiens
organism
Homo sapiens
disease staging
3C1
sex
female
scientific_name
Homo sapiens
common name
human
organism part
blood plasma
clinical information
developmental stage
adult
fraction
exosome
isolate
not applicable
ENA-FIRST-PUBLIC
2023-08-13T00:19:08Z
ENA-LAST-UPDATE
2023-08-13T00:19:08Z
age
58
ENA-FIRST-PUBLIC
2023-08-13
ENA-LAST-UPDATE
2023-08-13