Rna extraction kit

Total RNA was extracted with an appropriate RNA Extraction Kit (Promega, United States) and reverse-transcribed employing random primers and a Reverse Transcription Kit (Promega, United States). Then, the expression levels of target RNAs were evaluated using a Step One Plus Real-Time PCR System and SYBR Green Master Mix (7300R, ABI, United States). GAPDH was employed as an endogenous control, and the fold change was computed using the 2^−ΔΔCT technique (Mo et al., 2021 (link)). Primer sequences were designed by Sangon Biotech (Table 1).

Total RNA was extracted from seedlings grown under dark conditions at 2 d after gemination (DAG) using an RNA Extraction Kit (Promega, USA). A total of 3.0 µg of RNA was used to generate sequencing libraries using the NEBNext Ultra RNA Library Prep Kit following the manufacturer’s instructions (NEB, USA), and index codes were added to attribute sequences to each sample. Solexa sequencing was performed as a commercial service at Novogene (http://www.novogene.com/) with an Illumina HiSeq 2000 sequencer. The sequencing data were deposited in the NCBI’s Sequence Read Archive (SRA; https://www.ncbi.nlm.nih.gov/sra) under accession number SRP125848. Low-quality bases (Q<20) at the ends of the sequencing reads were trimmed using the SolexaQA software (Cox et al., 2010 (link)) (ver. 1.10, parameters: –b h 20); our RNA-seq data are shown in Supplementary Table S2. After trimming, all reads were mapped to the Arabidopsis genome (TAIR9; www.arabidopsis.org) using the TopHat software (Trapnell et al., 2009 (link)). Read counts were generated using HTSeq in union mode. Differentially expressed genes (DEGs) between samples were defined by Deseq (Anders and Huber, 2010 (link)), using a fold-change >2 and adjusted P-value <0.05.

Plants underwent one of four treatments: Control (CK), Melatonin (MT), Cold, and Melatonin + Cold (MT-C). Eight independent mRNA libraries with two biological replicates for each treatment were sequenced by a service provider Gene Denovo Co. (Guangzhou, China). After total RNA was extracted using the RNA extraction kit (Promega, USA), mRNA was enriched by oligo(dT) beads and then cleaved into short fragments using fragmentation buffer and reverse-transcribed into cDNA with random primers. Second-strand cDNA were synthesized by DNA polymerase I, RNase H, dNTP and buffer. Then the cDNA fragments were purified with QiaQuick PCR extraction kit, end repaired, poly (A) added, and ligated to Illumina sequencing adapter. The ligation products were size selected by agarose gel electrophoresis; PCR amplified, and sequenced using Illumina HiSeq^TM 2500. The sequences have been deposited into the NCBI Sequence Read Archive database (SRP078211, SRA438977).

Isolation of total RNA from treated samples was performed using an RNA extraction kit (Promega, Madison, WI, USA). The cDNA synthesis was performed with total RNA (2 μg) reverse transcribed using All-In-One RT MasterMix (Applied Biological Materials, Zhenjiang, China). RT-PCR analysis was conducted using 2 × T5 Super PCR Mix (Tsingke, Beijing, China) and Taq Master MixTaq mix (Vazyme Biotech, Nanjing, China). All primers used in this study are listed in Supplementary Table S2. Quantification for gel intensity was carried out using Image J software (https://imagej.nih.gov/ij/).

Total RNA in the hypothalamus of rats and PC12 cell lysates were extracted according to the RNA Extraction Kit (LS1040, Promega). Prime Script RT Reagent Kit (Takara, Shiga, Japan) was used to generate cDNA from mRNA. Real-time PCR was performed using the SYBR-Green Real-time PCR Kit (Takara) and a CFX96 real-time PCR detection system (Bio-Rad, USA). The mRNA expressions were calculated and analyzed by 2^−ΔΔCT formula and normalized to GAPDH.