GSM4585125: L37d-1C; Helianthus annuus; ncRNA-Seq
Ethanol precipitation protocol and CTAB-PBIOZOL reagent was used for the purification of total RNA from the plant tissue according to the manual instructions. Grind tissue samples about 80mg with liquid nitrogen into powder and transfer the powder samples in 1.5ml of preheated 65°C CTAB-pBIOZOL reagents. The samples were incubated by Thermo mixer for 15min at 65°C to permit the complete dissociation of nucleoprotein complexes. After centrifuge at 12000×g for 5min at 4°C, the supernatant was added 400ul of chloroform per 1.5ml of CTAB-pBIOZOL reagent and was centrifuged at 12000×g for 10min at 4°C. The supernatant was transferred to a new 2.0ml tube that added 700ul acidic phenol and 200ul chloroform followed by centrifuging 12000×g for 10 min at 4°C. The aqueous phase was added equal volume of aqueous phase of chloroform and centrifuged at 12000×g for 10 min at 4°C. The supernatant was added equal volume of supernatant of isopropyl alcohol and placed at -20°C for 2 hours for precipitation. After that, the mix was centrifuged at 12000×g for 20min at 4°C and then removed the supernatant. After washed with 1ml of 75% ethanol, the RNA pellet was air-dried in the biosafety cabinet and was dissolved by adding 50µL of DEPC-treated water. Subsequently, total RNA was qualified and quantified using a Nano Drop and Agilent 2100 bioanalyzer (Thermo Fisher Scientific, MA, USA). RNA libraries were prepared for sequencing using standard BGI500 protocols Library was prepared with 1 μg total RNA for each sample. Total RNA was purified by  electrophoretic separation on a 15% urea denaturing polyacrylamide gel electrophoresis  (PAGE) gel and small RNA regions corresponding to the 18–30 nt bands in the marker  lane (14-30 ssRNA Ladder Marker, TAKARA) were excised and recovered. Then the  18–30 nt small RNAs were ligated to adenylated 3' adapters annealed to unique  molecular identifiers (UMI), followed by the ligation of 5'adapters. The adapter-ligated  small RNAs were subsequently transcribed into cDNA by SuperScript II Reverse  Transcriptase (Invitrogen, USA) and then several rounds of PCR amplification with  PCR Primer Cocktail and PCR Mix were performed to enrich the cDNA fragments. The  PCR products were selected by agarose gel electrophoresis with target fragments  110~130 bp, and then purified by QIAquick Gel Extraction Kit (QIAGEN, Valencia,  CA). The library was quality and quantitated in two methods: check the distribution of  the fragments size using the Agilent 2100 bioanalyzer, and quantify the library using  real-time quantitative PCR (QPCR) (TaqMan Probe). The final ligation PCR products  were sequenced using the BGISEQ-500 platform (BGI-Shenzhen, China).