GSM5711962: Cell-D2; Rattus norvegicus; miRNA-Seq
MVs were isolated from chondrocyte cultures by differential centrifugation of the trypsin digested cell layer (briefly: after trypsinization - 0.25% Trypsin-EDTA - for 10 min, 1xDPBS containing 10% exosome-free FBS was added to stop the reaction. The MVs were isolated by sequential centrifugation at 500 g for 5min, 24,000 g for 20 min and 100,000 g for 70 min. The MV pellets were washed with 0.9% NaCl and recentrifuged at 100,000 g for 70 min.) NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs) RiboZero Magnetic Gold Kit (Human/Mouse/Rat) (Epicentre, an Illumina Company) NEB Multiplex Small RNA Library Prep Set for Illumina 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.