GSM3589701: TruSeq_PNK; synthetic construct; miRNA-Seq
10 femtomoles of the synthetic, equimolar pool were taken from previously-frozen aliquots for each library. The aliquots were either directly used in library construction, or pre-treated with T4 PNK. To perform phospho-small RNA-seq, synthetic and plasma RNA samples were pretreated with T4 polynucleotide kinase (NEB, Ipswich, MA, US) using an RNA input of 7 ul in a final volume reaction of 10 ul and following the manufacturer's instructions. After the enzymatic treatment, biological RNA samples were purified by performing sequential washes in silica columns (Zymo, Irvine, CA, US): (i) 900 ul of buffer RWT (Qiagen, Hilden, Germany); (ii) 900 ul of buffer RPE (Qiagen, Hilden, Germany); (iii) 900 ul of ethanol 200 proof, molecular biology grade (Fisher Scientific, Waltham, MA, US) and; (iv) 900 ul of 80% ethanol. Standard ligation-based small RNA libraries were prepared using the TruSeq small RNA kit (Illumina, San Diego, CA, US) according to the manufacturer instructions. Size selection was performed using pre-cast 6% acrylamide gels (Invitrogen, Waltham, MA, US) including all products from 140-200 bp plus any additional visible bands of greater size.