Urinary exosomal microRNA profile of diabetic rat through small RNA sequencing
Small RNA libraries for sequencing were constructed according to the Illumina TruSeq Small RNA library protocol outlined in “TruSeq Small RNA Sample Preparation Guide” (Part # 15004197; Rev. E; February 2013). ~200ng of RNA sample enriched for small RNAwas used as the starting material. Briefly, 3’ adaptors were ligated to the specific 3’OH group of small RNA followed by 5’ adaptor ligation. The ligated products were reverse transcribed with Superscript III Reverse transcriptase by priming with Reverse transcriptase primers. The cDNA was enriched by PCR (15 cycles) and cleaned using Polyacrylamide gel. The library was size selected in the range of 140 – 160 bp followed by overnight gel elution and salt precipitation using glycogen, 3M Sodium Acetate and absolute ethanol. The precipitate was re-suspended in Resuspension buffer.