Modban: The original protocol, as described by Lau et al.1, was followed. We used as input the 15-40 nt fraction of 1 μg of small RNA isolated using the miRVana kit. A preadenylated modban adapter was ligated to the to the RNA in the absence of ATP. The 30-50 nt fraction was excised from a 15% denaturing polyacrylamide gel. After elution in 0.3M NaCL and EtOH precipitation, the 5` linker was ligated in the presence of ATP. The 70-90 nt fraction was gel-excised from a 10% denaturing polyacrylamide gel, eluted and precipitated. Reverse-transcription was performed with the modban RT primer and 15 cycles of PCR were performed to amplify the library. 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.
