seeds under cold treatment for 12 h, replicate 1
Total RNA was initially fractionated on a 15% urea-PAGE gel electrophoresis and a band corresponding to small RNAs (18  30 bp) were excised. After small RNAs were extracted by centrifugation, they were ligated with the adenylated 3 adapter. Then, RT primer with barcode was used to anneal the 3 adenylated adapter, followed by the ligation of 5 adapter and the reverse transcript reaction. After first strand cDNA synthesis, we amplified the product by 15 cycles and carried out another size selection (103  115 bp) from the gel. After the gel purification, the PCR product was quantified by Qubit (Invitrogen) and a single strand DNA circle (ssDNA circle), which gave the final miRNA library, was made by pooling multiple samples together. For small RNA sequencing, we first generated the DNA nanoballs (DNBs) with ssDNA circle by rolling circle replication (RCR) to enlarge the fluorescent signals at the sequencing process. Then, the DNBs were loaded into the patterned nanoarrays and single-end read of 50 bp were read through on the BGISEQ-500 platform.