5 protocols
AccessionType
feature_extraction
Only 20-21 nt sequences with high quality were collected for subsequent analysis. The raw sequences were first normalized to “reads per 10 million” (RP10M). The distinct reads that perfectly matched soybean cDNA sequences remained. The 15 nt of sequence upstream and downstream of the 5’ end of matched reads were extracted to constitute 30-nt sequence tags for searching corresponding miRNA. The CleaveLand pipeline was used to align the 30 nt sequence to soybean known miRNAs from miRBase and our newly identified miRNAs. All alignments with scores up to 7 and no mismatches at the cleavage site (between the 10th and 11th nucleotides) were considered candidate targets.
feature_extraction
The raw data was preprocessed by Fastx-toolkit pipeline to remove low quality reads and clip adapter sequence. As to small RNA library, small RNAs ranged from 18-25 nt were collected and mapped to the soybean genome using SOAP2. The unique RNA sequences that perfectly matched the genome were subjected to the subsequent analysis. RNA reads, showing identical sequences to the known miRNAs from miRBase database, were picked up as the miRNA dataset of soybean. Sequences matching noncoding rRNA, tRNA, snRNA and snoRNA at Rfam database were removed. Reads overlapped with exons of protein-coding genes were excluded to avoid mRNA contamination. The remaining sequences were considered for prediction to find new miRNAs processed data file: 1st column: length of small RNA 2nd column: the counts of small RNA by solexa sequencing, 3rd column: unique sequence of small RNA
grow
Soybean seeds of (Glycine max) cultivar Heinong44 were directly planted in the Experimental Station of Institute of Genetics and Developmental Biology, Chinese Academy of Sciences in Beijing on May. Soybean seeds of 15 days after flowering (DAF) were collected
nucleic acid library construction protocol
By polyacrylamide gel electrophoresis, the small RNAs (~17-27 nt) were purified from 100 μg of total RNA and ligated to a 5’ RNA adapter and a 3’ RNA adapter. Reverse transcriptase reaction followed by low cycle PCR was performed to obtain sufficient product for SBS sequencing. PCR products were collected by gel purification and sequenced by Solexa technology.
nucleic acid library construction protocol
In brief, poly(A) RNA was extracted from 200 μg of total RNA using the Oligotex kit (Qiagen). A 5’ RNA adapter containing a MmeI recognition site was ligated to the poly(A) RNA possessing a 5’-phosphate, by T4 RNA ligase (Ambion), and the ligated products were repurified using the Oligotex kit. Five PCR cycles were then performed to amplify the reverse transcription products. The PCR products were digested with MmeI and ligated to a 3’ double DNA adapter. The ligation products were amplified by 20 PCR cycles and gel-purified for SBS sequencing.