polyA: The 15-40 nt fraction derived from 1 μg small RNA (<200 nt) was polyadenylated, purified and treated with periodic acid (as in 2) for 3` termination of the poly(A) tail. After ligation of the 5` linker, the >70 nt fraction was excised from 10% denaturing polyacrylamide gel (for replicate 1) or this step was omitted (replicate 2). Reverse-transcription was performed with the poly(A) RT primer, followed by 15 cycli of PCR (replicate 1) or 1/10 of the RT reaction was amplified by 17 cycles of PCR (replicate 2). We produced 454 compatible libraries with ~0.3 ng of each library. Primers used were barcoded primers (454solid_bc) in the forward orientation and the 454solid_reverse primer in the reverse orientation. 8 rounds of PCR were performed for each library as the PCR in the modban and poly(A) library preparation, with the exception that all reagents were 5x concentrated. The libraries, each carrying a unique barcode in the 5` adapter, were equimolarly mixed and submitted for 454 sequencing. 454solid_bc: 5` GCC TCC CTC GCG CCA TCA G barcode C CAC TAC GCC TCC GCT TTC CTC TCT ATG 3` Barcodes:Bc1 GCTA;Bc2 CGTA;Bc3 CGAT;Bc4 GCAT;Bc5 TAGC;Bc6 ATGC
Library strategy
RNA-Seq
Library source
transcriptomic
Library selection
size fractionation
Instrument model
454 GS FLX
Description
none
Data processing
Sequencing reads were mapped to the known miRNA and miRNA* species in miRBase v11.0 using SHRiMP. Reads with 3 or less errors were included in the further analysis.
