title: GSM5258574: Blank_3; Homo sapiens; miRNA-Seq
library_construction_protocol: Cell culture supernatant was collected from the control group and the xanthatin group. Supernatant were collected by differential centrifugation (500xg at 4℃ for 5min, 2000xg at 4℃ for 30min, 10000xg at 4℃ for 60min). Filtrated with a 0.22um sterile filter, added into the ultra-high speed centrifuge tube, centrifuged at 4℃ for 70min at 120000xg and discarded the supernatant. Finally, depending on the amount of precipitation, 200-400 L of pre-cooled sterile PBS was added for resuspended suspension, which was high purity EVs. Total RNA was extracted by TRIzol reagent, and the RNA molecules in a size range of 18–30 nt were enriched by poly-acrylamide gel electrophoresis. The 3'adapters were added and the 36–44 nt RNAs were enriched. The 5'adapters were then ligated to the RNAs as well. The ligation products were reverse-transcribed by PCR amplification, and the 140–160 bp size PCR products were enriched to generate a cDNA library. The cDNA fragments were purified with PCR extraction kit, poly(A) added and ligated to Illumina sequencing adapters