Instrument: Illumina HiSeq 2000
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: size fractionation
Layout: SINGLE
Construction protocol: miRNA-Seq libraries are performed from at least 1µg of extracted total RNA with a RIN greater than 7. Before starting, total RNAs are purified with miRNeasy kit wich allows the selection of the small RNA fraction less than 100b. From these samples enriched in small RNAs, libraries are performed according to the protocol published by François Vigneault : Curr Protoc Hum Genet. 2012 Apr;Chapter 11:Unit 11.12.1-10. "High-throughput multiplex sequencing of miRNA"; Vigneault F, Ter-Ovanesyan D, Alon S, Eminaga S, C Christodoulou D, Seidman JG, Eisenberg E, M Church G. miRNA-Seq libraries are performed from at least 1ug of extracted total RNA with a RIN greater than 7. Before starting, total RNAs are purified with miRNeasy kit wich allows the selection of the small RNA fraction less than 100b. From these samples enriched in small RNAs, libraries are performed according to the protocol published by François Vigneault : Curr Protoc Hum Genet. 2012 Apr;Chapter 11:Unit 11.12.1-10. High-throughput multiplex sequencing of miRNA; Vigneault F, Ter-Ovanesyan D, Alon S, Eminaga S, C Christodoulou D, Seidman JG, Eisenberg E, M Church G. Extraction with Qiacube-miRNeasy 96 Mini (Qiagen) kits; 1 to 10 ug per sample, concentration in range 70-500 ng/uL, volume range 15 to 50 uL, BioAnalyzer profile RIN > 7; Ligation, amplification , quantification and purification according to manufacturer's instructions, with the following details: First, a 3 prime adenylated DNA adaptor is ligated to the enriched sample in the absence of ATP preventing the self-ligation of miRNAs.Then a 5 prime RNA adaptor is ligated in the presence of ATP at the other end of the miRNAs.The RT primer complementary of the 3 prime adaptor is added at this stage with which it will form a duplex thereby reducing the ligation between adaptors. A reverse transcription is therefore perfomed from the RT primer and finally these captured miRNAs are amplified by PCR with primers complementary to the 3 prime and 5 prime adaptors. During this PCR a specific barcode is incorporated allowing individualisation of each library.Each PCR is loaded on the Fragment Analyzer (AATI) for a precise quantification of each miRNA peak of interest.Based on these results a equimolar pool of about ten of different samples are performed. Finally the pooled PCR product is loaded on PAGE in order to excise the band of miRNA that is extracted and purified on a Qiagen MinElute column. Pool Concentration between 0.97 and 4.84 nM per uL; purification quantity is 3500 ng