GSM7469185: Serum-derived exosomes, DM-CMD, 1; Homo sapiens; miRNA-Seq
The Peripheral blood was subjected to 3000g Centrifuge for 15min and the upper serum was obtained. We added an appropriate amount of Exosome Precipitation Solution (System Biosciences, USA) at a ratio of 63ul of solution per 250ul of serum. After pre-cooling at 4°C for 30min, the mixture was centrifuged at 1500g for 30min to precipitate exosomes. The total RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, CA, USA) following standard procedures.The purity and integrity of the RNA were assessed using a ND-1000 Spectrophotometer (NanoDrop). The raw reads from small RNA sequencing are in fastq format, and the linker sequence is removed using cutadapt. Use fastx_toolkit (version 0.0.13) software to perform Q20 quality control on sequences, retaining sequences with Q20 of 80 and above. NGSQCToolkit (version 2.3.2) was then used to filter out reads containing N-bases. The result is high-quality clean reads that can be used for subsequent analysis. Statistics were performed on the length distribution of clean reads to initially assess the distribution of small RNAs in the sample. Using blastn software, clean reads are compared with Rfam (version 10.0) databases to annotate sequences such as rRNA, snRNA, snoRNA, tRNA, etc. Using the RepeatMasker software, the filtered sequences are compared to the repeat database to identify possible repeats. Small RNA sequences on unannotated devices were predicted using Mirdeep2 software for new miRNAs and secondary structures of miRNAs were predicted using RNAfold software. The miRNA expression calculation uses TPM (transcript per million) to calculate the metric, TPM = number of reads per miRNA / total sample alignment read number x106.