GSM9363846: miRNA-Seq, PA1-Dicer mutation S1344L homozygous; Homo sapiens; miRNA-Seq
The Qiagen miRNeasy kit was used to isolate the total RNA fraction Libraries were prepared from 500 ng of each total RNA sample using the QIAseq miRNA library kit 96 (QIAGEN, #331505) and the QIAseq miRNA 96 Index Kit IL UDI-E (#331655) according to the provided protocol. Mature miRNAs possess both a 3' hydroxyl group and a 5' phosphate. A pre-adenylated DNA adapter was ligated to the 3' ends of all miRNAs and then an RNA adapter was ligated to the 5' end of mature miRNAs. After both ligations, the ligated miRNAs were reverse-transcribed into cDNA. The reverse transcription primer contains an integrated UMI and binds to a region of the 3' adapter where it can facilitate conversion of the ligated miRNAs into cDNA while assigning a UMI to every miRNA molecule. During this step a universal sequence is also added that is recognized by the sample indexing primers during library amplification. Libraries were purified using QIASeq miRNA NGS Beads (QMN beads). Purified libraries were then amplified for 13 cycles of PCR with unique 10bp dual index (UDI) sequences to allow multiplexing on a single sequencing run. Amplified indexed libraries were purified and size selected with QMN beads to enrich for library fragments from miRNA.