title: GSM4020180: piRNA_Day_7_Chondro_donor#3_replicate#2; Homo sapiens; ncRNA-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 piRNA-sequencing library, which included the following steps: 1) 3'-adapter ligation; 2) 5'-adapter ligation; 3) cDNA synthesis; 4) PCR amplification; 5) size selection of ~142-153bp PCR amplified fragments (corresponding to ~24-35 nt small RNAs). The libraries are qualified and quantified using Agilent BioAnalyzer2100. 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 follow the manufacturer's instructions.