miRNA-seq of Mesenchymal Stem Cells: control
RNA was extracted from MSCs using theQIAGEN miRNeasy kit. Total amounts and integrity of RNA were assessed using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, CA, USA). A total of 1 g total RNA per sample was used as input material for the small RNA library preparation. Briefly, 3 and 5 adaptors were ligated to 3 and 5 end of small RNA, respectively. Then the first strand cDNA was synthesized after hybridization with reverse transcription primer. The double-stranded cDNA library was generated through PCR enrichment. After purification and size selection, libraries with insertions between 18~40 bp were ready for sequencing on Illumina sequencing with SE50. The library was checked with Qubit and real-time PCR for quantification and bioanalyzer for size distribution detection. Quantified libraries were pooled and sequenced on Illumina platforms, according to effective library concentration and data amount required.