GSM1581811: PorcineEndometrium_C16_17; Sus scrofa; miRNA-Seq
Total RNA containing small RNAs was extracted using a mirVana miRNA isolation kit (Life Technologies, USA) according to the manufacturer’s protocol. The protocol includes sample disruption in lysis buffer, acid phenol:chloroform extraction and an on-column RNA isolation. Quality and quantity of RNA was determined using UV spectrophotometry (NanoDrop 1000). RNA with RNA Integrity number higher than 7 (Agilent 2100 Bioanalyzer, Agilent Technologies, Santa Clara, CA) was used for small RNA libraries preparation. 5µg of total RNA form each sample was used for sequencing with NEXTflex Small RNA Sequencing Kit (BiooScientific, USA) according to supplier’s protocol. 3’ and 5’ adapters were ligated to the RNA using AIR ligase and T4 RNA ligase, respectively. During the 5’ ligation, NEXTflex RT primers were also incorporated to the RNA. After 3’ and 5’ primer ligation, the 140 bp RNAs were purified by Clean & Concentrator 5 Kit (Zymo Research, Irvine, CA, USA) to eliminate unligated products. PCR amplification of the ligated products was accomplished by using 1-12 primer barcodes (NEXTflex Small RNA Barcode Primers, BiooScientific) and DuroTaq 5x PCR Master Mix (BiooScientific) for multiplexing. Concentration of each library was determined with Agilent High Sensitivity DNA Kit using Agilent 2100 Bioanalyzer (Agilent Technologies, Waldbronn, Germany). Final concentration of 30 nmol/l of each library was used to prepare 3 pools for multiplex analysis. Size fractioning of the pooled cDNA was done by 2% agarose gel electrophoresis in the presence of 0.5 ng/µl of ethidium bromide. Small RNA fraction with ligated adaptors ranging around 140-160 bp was excised from the gel with X-TRACTA II (Biozym Scientific GmbH, Hessisch Oldendorf, Germany), purified (Ultrafree-DA, Millipore, Billerica, MA, USA) and sequenced.
GEO Accession
GSM1581811