SAMD00009450
External Id
RIKEN Omics Science Center, Yokohama Institute, RIKEN
INSDC center name
2014-03-04T00:00:00Z
INSDC first public
2015-10-13T01:01:53Z
INSDC last update
DRS002065
INSDC secondary accession
live
INSDC status
Generic
NCBI submission model
Generic.1.0
NCBI submission package
DRS002065
SRA accession
Homo sapiens
http://purl.obolibrary.org/obo/NCBITaxon_9606
organism
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.751Ã¢ÂÂ755), with template-switching (Ko &amp; Y. Lee 2006, Journal of Microbiological Methods, 64(3), pp.297Ã¢ÂÂ304) to enrich for capped RNAs (Plessy et al. 2010, Nature Methods, 7(7), pp.528Ã¢ÂÂ534 ). 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 either with three ribocytosines (5'-TAGTCGAACTGAAGGTCTCCAXXXrCrCrC-3') or three riboguanosines (5'-TAGTCGAACTGAAGGTCTCCAXXXrGrGrG-3') where is XXX is barcode'. The cDNAs were amplified using 12Ã¢ÂÂ15 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 100Ã¢ÂÂbp 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)
sample comment
DRS002065
sample name
S92: template-switched short RNA fractions libraries (G/C version)
title