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DRS002064 |
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Small RNA was isolated from THP-1 cells using the mirVana kit (Ambion) according to manufacturer's instructions. In summary, we combined the dimer eliminators from (Kawano et al. 2010, BioTechniques, 49(4), pp.751â755), with template-switching (Ko & Y. Lee 2006, Journal of Microbiological Methods, 64(3), pp.297â304) to enrich for capped RNAs (Plessy et al. 2010, Nature Methods, 7(7), pp.528â534 ). In brief, adenylated 3' adapters adenylated in 5' and blocked in 3' (5'-AppATCTCGTATGCCGTCTTCTGCTTG-idT-3') were ligated to 3' ends of 3'-OH small RNAs using a truncated T4 RNA ligase 2 enzyme (NEB). cDNA was primed using a oligonucleotide complementary to the 3' adaptor in the presence of dimer eliminator and template-switching oligonucleotides ending with three random ribonucleotides 5'-TAGTCGAACTGAAGGTCTCCAXXXrNrNrN-3 where is XXX is barcode'. The cDNAs were amplified using 12â15 PCR cycles using a forward primer targeting the 5' adaptor and extending it with additional sequence for bridge PCR, and a reverse primer targeting the 3' adaptor (which is ready for bridge PCR). The amplified cDNA library was run on a 6 % polyacrylamide gel and the 100 bp to 300 bp range containing cDNAs up to 200 nt-long inserts cut in three fractions according to the insert size: 1-50 nt, 50-150 nt and 1-200 nt. The fractionated libraries were then extracted using standard extraction protocols (Kawano et al. 2010). |
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