title: GSM3476837: control, replicate 2; Homo sapiens; miRNA-Seq
library_construction_protocol: miRNA was isolated using the miRNeasy Mini Ki from Qiagen according to the manufacturer's instructions; miRNA quantity and quality were checked by Agilent Bioanlyzer Libraries were prepared using the QIAseq miRNA Library Kit (QIAGEN Cat. No. 331502), following manufacturer's instructions.  a. 3' ligation: 500 ng of RNA samples (in a volume of 5 µl) were combine with 1 µl 3' adapter, 1 µl RI, 1 µl 3' ligase, 2 µl 3' buffer and 10 µl ligation activator. The reaction mix was incubated for 1 hour at 28°C, then 20 minutes at 65°C. b. 5' ligation:  To the 3' ligation reaction, add 15 µl H2O, 2 µl 5' buffer, 1 µl RI, 1 µl 5' ligase and 1 µl 5' adapter. The reaction mix was incubated for 30 minutes at 28°C, then 20 minutes at 65°C. c. Reverse Transcription: Add 2 µl RT initiator to the reaction, incubate 2 minutes each at 75°C, 70°C, 65°C, 60°C, 55°C, then 5 minutes at 37°C, and 5 minutes at 25°C. Add 2 µl RT primer, 2 µl H2O, 12 µl RT buffer, 1 µl RI and 1 µl RT enzyme. The reaction mix was incubated for 1 hour at 50°C, then 15 minutes at 70°C.  d. cDNA cleanup: Reactions were cleaned up with magnetic beads (QIAseq miRNA NGS beads). cDNA was eluted in 15 µl H2O.  e. Library Amplification:  cDNA was amplified in a thermal cycler for 13 cycles at the following temperature 95°C for 15 sec, 60 °C for 30 sec, 72 °C for 15 sec, 72°C for 2 minutes and hold for 4°C. Sample and Barcode Information: Samples were barcoded with QIAseq miRNA NGS ILM IPD primers.
attributes.tag: GEO Accession
attributes.value: GSM3476837