NextSeq 550 sequencing; RNA-seq of human plasma microRNAs in Alzheimer Disease
RNA-seq of human plasma microRNAs in Alzheimer Disease
Plasma samples were obtained from all participants. They were stored at -80 ºC 700 µL QIAzol lysis reagent were added to 200 µL plasma, vortexed and incubated at 20-25 ºC for 5 min. After that, 140 µL of chloroform were added and incubated at room temperature for 3 min. Then, it was centrifuged (15min, 12000g, 4 ºC), and the aqueous phase transferred to a new tube containing 525 µL ethanol. The mixture was transferred into an RNeasy MinElute spin column, centrifuged (10000g, 30s, RT). Column was washed with 700 µL buffer RWT and twice with 500 µL buffer RPE. Membrane was dried by centrifugation (10000g, 90s) and elution was carried out in 15 µL RNase-free water centrifuging (1 min, 13000g) the smallRNA molecules are first ligated to the 3'- 4N adenylated adapters, taking advantage of the phosphate group at their terminal end, which allows these molecules to be exclusively selected. Second the 5'-4N adapters are ligated. After ligation of the adapters, the reverse transcription of the molecule to cDNA is carried out. The generated cDNA fragments are amplified and indexed by PCR using different barcode primers for each sample. Finally a purification with size selection is carried out. The quality control and concentration of the libraries are verified with the Agilent Technologies 2100 bioanalyzer using highly sensitive DNA chips. Subsequently, an equimolecular pool of each library is prepared for sequencing on the NGS NextSeq 550 platform (Illumina, San Diego, CA, USA) by single read sequencing of 50 cycles (1x50 bp).