title: GSM3711392: miRNA day_0_2D - donor#1 - replicate#1; Homo sapiens; miRNA-Seq
library_construction_protocol: Total RNA was isolated from day 0 2D and 3D samples collected in TRIreagent following manufacturer's instructions. Briefly, 100 µl of 1-Bromo-3-chloropropane (Sigma-Aldrich, St. Louis, MO, USA) were added to TRIreagent, mixed thoroughly and the samples were centrifuged at 12000 g for 15 min at 4°C. The recovered aqueous phase was mixed with 500 µl of 2-propanol (Sigma-Aldrich) and precipitation of RNA was carried out overnight at -20°C for enhanced recovery of small RNA species. RNA pellet was then washed with 75% EtOH (Sigma-Aldrich) and resuspended in 20 µl of DEPC-treated H2O. Total RNA concentration was measured with NanoDrop 1000 (Thermo Fisher) and purity was assessed by evaluation of the A260/280 and A260/230 ratios. Total RNA of each sample was used to prepare the miRNA sequencing library, which included the following steps: 1) 3'-adaptor ligation; 2) 5'-adaptor ligation; 3) cDNA synthesis; 4) PCR amplification; 5) size selection of 135~155 bp PCR amplified fragments (corresponding to 15~35 nt small RNAs). The libraries were denatured as single-stranded DNA molecules, captured on Illumina flow cells, amplified in situ as clusters and finally sequenced for 51 cycles on Illumina NextSeq per the manufacturer's instructions. Agilent 2100 Bioanalyzer was used for assessment of the quality of sequencing library. Library concentration was determined by qPCR method.