GSM5049161: miRNAs-M10_2T; Homo sapiens; miRNA-Seq
Total RNA was extracted from pregnant women plasma samples (2 mL) at 1st, 2nd and 3rd trimester (same women for each trimester) using TRI Reagent Solution (Ambion), in a 3:1 sample volume ratio. Samples were mixed and incubated for 5 minutes at room temperature and centrifuged at 12,000 x g for 10 minutes at 4 °C. The remaining aqueous phase was removed and mixed with chloroform (0.2 mL per mL of TRI Reagent), the new aqueous phase was transferred to fresh tubes and isopropanol (0.5 mL per mL of TRI Reagent) was added, mixed, and incubated overnight at -75 °C. After incubation samples were centrifuged at 12,000 x g for 15 minutes at 4 °C. RNA pellet was resuspended in 75% ethanol and centrifuged at 7,500 x g at 4 °C for 5 minutes. RNA pellets were resuspended in 10 uL of RNase-free water and stored at -75 °C. Library construction for small RNA sequencing analysis was performed with the NextFLEX small RNA-Seq kit v3 (Bioo Scientific), bands in an acrylamide gel, ranging from 50 to 150 bp were processed. Libraries were sequenced for 150 cycles, single-end, on an Illumina NextSeq 500.