miRNA-seq of exosomes derived from peripheral blood
Total RNA was isolated and purified following the manufacturers procedure. The RNA amount and purity of each sample was evaluated using NanoDrop ND-1000(NanoDrop, Wilmington, DE, USA). The RNA fragment integrity was evaluated through Bioanalyzer 2100 (Agilent,CA,USA). The obtained total RNA were denaturated at 70 for 2 min with DNA 3 Adapters , and then mixed with T4 RNA Ligase 2, truncated K227Q (NEB, M0351L, USA) at room temperature. The 3 adapter ligation reaction was performed at 16 more than 8 hours. The rest of 3Adapters were removed by RTP at 37 for 30 min. The 5 adapters and the products from last step were ligation with T4 RNA Ligase 1 (NEB,M0204L,USA) at 37 for 60 min following with reverse-transcription reaction carried by SuperScript II Reverse Transcriptase (Thermo,18064014,USA) at 50 for 60 min, and at 80  for 10 min. The cDNA products were amplified using Phusion High-Fidelity DNA Polymerase (NEB,M0530L,USA). Specifically, the denaturation temperature was 98 for 30 sec with an annealing temperature of 60 for 30 sec, and the extension was carried out for 15 sec at 72 for 10-16 cycles. The final 5 minute extension was then done at 72.The PCR products were purified and enriched through PAGE electrophoresis. The sequencing strategy was Single-end 50 bp for Illumina Hiseq 2500 following the vendors recommended protocol.
SUB15808802
NC-1-27