Assay summary for miRNA-seq_Mathavan_Lab_0001AS

Assay Type:
Mathavan Lab, NTU Medical School
Total RNA were extracted using mirVana™ miRNA Isolation Kit (AM1560, Life Technologies). Tissues were homogenised in 1.5 ml microfuge tube containing Lysis/Binding buffer provided in the mirVana™ miRNA Isolation using a hand held pestle. Total RNA containing small RNA were purified following the manufacturer protocol.Small RNA libraries were prepared for sequencing using TruSeq Small RNA Sample Preparation Kit ( RS-200-0012, Illumina, Inc.). Libraries were prepared according to manufacturer instructions. Briefly, 1µg of good quality Total RNA per sample was used as starting material. 5’ and 3’ RNA adapters were ligated to each RNA molecule before reverse transcription to create single stranded cDNA. The cDNA was then amplified with PCR using a common primer and a primer containing a unique index sequence. The resulting PCR reactions were electrophoresed on 6% Novex TBE PAGE Gel (Life Technologies) and bands corresponding to adapter-ligated constructs derived from 22-30 nucleotides small RNA fragments were excised from the gel. The small RNA were purified from the excised gel and validated on High Sensitivity DNA chips on a Bioanalyser (Agilent Technologies) before sequencing.
Library Preparation:
Total RNA
Added by User:
Date Added:
April 5, 2017, 5:54 a.m.
Last Edited:
Aug. 9, 2018, 2:20 a.m.