GSM6314931: LC2; Bos taurus; miRNA-Seq
Small RNA-Seq librairies were generated from NA to 200ng of total RNA (or from --- ng of purified small RNA fraction) using TruSeq Small RNA Library Prep Kit (Illumina, San Diego, CA), according to manufacturer's instructions. The protocol takes advantage of the natural structure common to most microRNA molecules that have a 3' hydroxyl group resulting from enzymatic cleavage by Dicer or other RNA processing enzymes. Briefly, in the first step, RNA adapters were sequentially ligated to each end of the RNA, first the 3' RNA adapter that is specifically modified to target microRNAs and other small RNAs, then the 5' RNA adapter. Small RNA ligated with 3' and 5' adapters were reverse transcribed and PCR amplified (30 sec at 98oC; [10 sec at 98oC, 30 sec at 60oC, 15 sec at 72oC] x 13 cycles; 10 min at 72oC) to create cDNA constructs. Amplified cDNA constructs of 140 to 160 nt were selectively isolated by acrylamide gel purification followed by ethanol precipitation.  The final cDNA libraries were checked for quality and quantified using capillary electrophoresis.