Rna extractor kit

Two-week-old seedlings were collected from 1/2× MS plates and used for RNA-seq with three biological replicates. The total RNA was extracted using an RNA extractor kit (Sangon, China) according to the manufacturer’s protocol, and treated with RNase-free DNase I to remove genomic DNA contamination. The quality of RNA was assessed using an RNA HS assay kit (Thermo Scientific), quantified with Qubit2.0 (Thermo Scientific). The qualified RNA was used for sequencing and library construction using VAHTSTM mRNA-seq V2 Library Prep Kit for Illumina® following the manufacturer’s recommendations. The sequencing of the library was performed on an Illumina HiSeq3000 system at Sangon biotech (China). After evaluating and filtering raw data using FastQC (version 0.11.2) and Trimmomatic (version 0.36), the clean data were mapped to the Arabidopsis genome (TAIR10) by hisat2 [58 (link)] and used to assemble transcripts and to calculate the expression of genes using StringTie [59 (link)]. The DEGs were analyzed by Deseq2 [60 (link)]. Genes were considered as significantly differentially expressed at a p-value < 0.05 and |FoldChange| > 2. The GO enrichment analyses and KEGG pathway functional enrichment analysis of the DEGs were performed using KOBAS (http://kobas.cbi.pku.edu.cn/anno_iden.php) [61 (link)].

As described by Sun et al.^{36 (link)}, T₁ seeds were surface-sterilized with 75% ethanol for 1 min and 10% NaClO for 10 min, and plated onto 1/2 MS plates with 50 mg/L kanamycin (Sigma, USA) for screening of transgenic plants, which were used to produce T₂ generation. The T₂ plants with 3:1 segregating-ratio to resistance/susceptibility of kanamycin were self-pollinated to generate T₃. The homozygous lines without segregation were collected from T₃, and were identified by PCR amplification using the primers (Table S4) for the specific fragments of AnAFP, AnAFPΔA, AnAFPΔK, AnAFPΔN and AnAFPΔS. The total RNA of every line was extracted by RNA extractor kit (Sangon, China), reacted with RNase-free DNase I, and used to reverse transcribed into cDNA using PrimeScript RT Reagent Kit (TaKaRa, Dalian). The ectopic expression of the transformed genes was identified by reverse transcription PCR (RT-PCR) using the above primers. The AtActin gene was amplified and used as reference. Five T₃ lines were planted in pots, and grown in green house at 22 ℃ and 60–70% relative humidity under a 10 h light/14 h dark photoperiod. One-month-old seedlings were used for heat-shock treatment at 46 °C for 3 h, and recovered for 2 weeks at 22 ℃, and investigated for phenotype.