GSM9488763: CE3; Homo sapiens; miRNA-Seq
1. Take the frozen serum samples out of the -80°C refrigerator, thaw them on ice, and centrifuge at 4000 rpm, 4°C for 20 minutes to further remove residual cell debris and impurities, and collect the supernatant. 2. Purify exosomes from the supernatant by size exclusion chromatography (SEC): first, equilibrate the SEC column with pre-cooled PBS (RNase-free) to ensure the column is free of impurities and air bubbles; then load the supernatant onto the column, elute with PBS at a constant flow rate, and collect elution fractions sequentially. 3. Conduct quality control on the collected fractions: take a small amount of each fraction for ultra-high-speed centrifugation (100000 rpm, 4°C for 70 minutes), observe whether exosome precipitates are formed; combine the fractions containing exosomes, and centrifuge again to concentrate exosomes, which are used for subsequent identification and RNA extraction. 1. Construct small RNA libraries using the Illumina TruSeq Small RNA Library Prep Kit, strictly following the kit instructions: ligate 3' and 5' adapters to the purified exosomal small RNA, perform reverse transcription to synthesize cDNA, and amplify the cDNA by PCR to obtain the small RNA library. 2. Qualify the constructed library: detect the library size (target size 140-160 bp) by agarose gel electrophoresis, and determine the library concentration and uniformity by quantitative PCR (qPCR). 3. Perform single-end 50 bp sequencing on the qualified libraries using the Illumina Hiseq 2000/2500 sequencing platform. During the sequencing process, set up quality control samples to monitor the sequencing quality (sequencing error rate < 1%, Q30 value > 85%).