Total RNA was isolated from leaves and flower buds using TRIZOL (Invitrogen) according to manufacturer’s instructions. Small RNAs of 15-100-nt in size were purified in a 15% polyacrylamide gel and ligated to the 5’ and 3’ RNA adaptors, respectively. The resulting RNA bands ranged from 55- to 140-nt. The ligated RNAs from each sample were reverse-transcriptase transcribed, and the first-stranded cDNAs were amplified using the primer pair that contains 2 specific nucleotides (out of 4) as a “barcode”. A. arenosa and F1 leaf libraries were sequenced separately, and the remaining eight libraries were combined in two pools, and an aliquot of pooled DNA was cloned and sequenced to determine the quality and representation of inserts. After the quality control was done, the pooled DNA was subjected to high-through pyrosequencing, producing a total of ~1.5-million reads in eight runs.
Library strategy
miRNA-Seq
Library source
transcriptomic
Library selection
size fractionation
Instrument model
454 GS
Description
progenitor species 5' adaptor sequence: GCCTCCCTCGCGCCATCAGtgatATCGTAGGCACCTGAAA raw data file(s): EJSVV8G01.sff, EJSVV8G02.sff, EJS6HNP01.sff, EJS6HNP02.sff, EJ4IYVT01.sff, EJ4IYVT02.sff
Data processing
The raw sequences were processed by identifying the barcodes, base call quality, and removing adaptor sequences. Each of the 10 libraries had a unique 4-nucleotide tag within the 5’-end adaptor sequences. After trimming adaptor sequences at both 5’ and 3’ ends, pyrosequenced cDNAs were aligned to full genomic sequences of A. thaliana. Sequences matching other cellular RNAs including pre-tRNAs, rRNAs, snoRNAs, and snRNAs were regarded as degradation products of other cellular RNAs and excluded for analysis. Also excluded were sequences matching genome sequences of mitochondria and chloroplasts. Remaining sequences 20-25-nt in length.
