Rnase free dnase 1 kit

For ROC22, the samples of control groups were labeled as S1–S3, and those of the treatment groups were labeled as S4–S6. For FN39, the samples of control groups were marked as S7–S9, and those of the treatment groups were marked as S10–S12. Total RNA was extracted from the above 12 samples using Trizol reagent (Invitrogen, Shanghai, China). Nanodrop, Qubit 2.0 and Agilent 2100 bioanalyzer were used to detect the purity, concentration and integrity of RNA samples. Before cDNA synthesis, the genomic DNA was removed according to the instructions of the RNase-Free DNase I kit (Promega, Fitchburg, WI, USA). Then the RNA was reversed into the first strand of cDNA referred to the instructions of Prime-Script™ RT Reagent Kit (TaKaRa, Dalian, China). The sequencing (HiSeq2500) and library construction of sugarcane small RNAs were commissioned by Biomarker Biotechnology Co., Ltd. (Beijing, China). The read length was 50 nt.

About 2.0 µg RNA from floral buds was treated with a RNase-free DNase I Kit (Promega, USA) in a 10 µl volume to remove genomic DNA contamination. The first-strand cDNA was synthesized with the oligo (dT)₁₈ primers using M-MLV cDNA synthesis kit (Invitrogen, China) following product instructions in a 20 µl volume. Full-length cDNA sequences of the involved genes were obtained by a routine RT-PCR method using gene-specific primers (Supplementary Table S1). Each amplified fragment was purified using the High Pure PCR Product Purification Kit (Roche, Switzerland). Purified fragments were ligated into the pEASY®-Blunt Cloning vector (TransGen Biotech, China) and transformed into Trans10 chemically competent cells (TransGen Biotech, China). Sequencing was performed by Taihe Biotech, China.