GSM5861413: lung cancer 4; Homo sapiens; miRNA-Seq
1.5ml of plasma was diluted in PBS to 10ml. The sample underwent differential centrifugation at 500×g for 5 min at 4 ℃ and 2000×g for 30 min at 4 ℃, followed by 10000×g for 45 min at 4 ℃. After that, the supernatant was filtered through a 0.45 um membrane filter and ultracentrifuged at 100000×g for 70 min at 4 ℃, then resuspended in PBS and underwent a second ultracentrifugation at 100000×g for 70 min at 4 ℃. The obtained exosomes were resuspended in PBS. The miRNAs were isolated from plasma-derived exosomes using TRIzol LS Reagent (Invitrogen) according to the manufacturer' s instruction. During RNA precipitation with isopropanol, glycogen was added for co-precipitation to improve the yield of RNA. The yield and quality of extracted RNA were evaluated by NanoDrop 1000 Spectrophotometer (NanoDrop Technologies, Delaware, USA). RNA samples were used to prepare the miRNA sequencing library, which included the following steps: (1) 3′-adaptor ligation, (2) 5′-adaptor ligation, (3) cDNA synthesis, (4) PCR amplification, and (5) size selection of ~135–155 bp PCR amplified fragments (corresponding to ~15–35 nt small RNAs). The sequencing library was determined by an Agilent 2100 Bioanalyzer using an Agilent DNA 1000 chip kit (part # 5067-1504, Agilent, Santa Clara, CA, USA). And then the libraries were denatured as single-stranded DNA molecules, captured on Illumina flow cells (Illumina), amplified in situ as clusters and finally sequenced for 50 cycles on an Illumina NextSeq per the manufacturer's instructions.