Mrna seq library prep kit for

Total RNA was extracted and evaluated using the previous method. The sequencing library was generated by using the mRNA-seq Library Prep Kit for Illumina (Vazyme Biotech, China), following the manufacturer's protocols. In brief, mRNA (1–4 μg) was separated by Capture Beads, then the isolation mRNA was fragmented with the Frag/Prime Buffer. The cDNA was synthesized using the first Strand Mix, and the second Strand/End Mix. The cDNA was connected with adapters, sorted by fragment, and enriched with PCR. The sequencing of libraries was performed by an Illumina HiSeq 2500 platform (USA).

Nine plants at bud stage in each group were randomly selected, three of which were pooled as a repeat and a total of three replicates were set for each group. The second leaf (the leaf under flag leaf) and root tissues were sampled for RNA-seq analysis using Illumina HiSeq 4000 platform. Three leaf (a) and root (b) samples were obtained from “X”, “Y” and “C”. Total RNA in each sample was purified using the cetyltrimethylammonium bromide (CTAB) method according to Thunyamada’s report [20 ]. Genomic DNA contamination were removed by DNase I (Takara, Tokyo, Japan). RNA concentration was determined using a NanoDrop ND-2100 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). Then, RNA sequencing library was constructed using a mRNA-seq Library Prep Kit for Illumina (Vazyme, Nanjing, Jiangsu, China). All the RNA sequencing libraries were then detected using a Qubit 2.0 fluorometer (Invitrogen, Carlsbad, CA, USA). In addition, the integrity of RNA was also determined by an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). All these libraries were then subjected to the Illumina HiSeq 4000 platform followed by 2 × 150 bp paired-end sequencing.

RNA was isolated from UC-MSCs treatment with or without TC14012 (30 μM) after 24 h by performed with the Direct-zol RNA MiniPrep Kit (Zymo Research). The RNA sequencing libraries were prepared using the mRNAseq Library Prep Kit for Illumina® (NEB England BioLabs). An Illumina Novaseq 6000 was used to sequence fragmented and randomly primed paired-end library pairs. Three biological replicates were sequenced for each group. The analysis of gene expression enrichment, heat maps, and gene set enrichment analysis were generated using clusterProfiler.