Hiseq ten system

Total RNA from the root, stem, and leaf of 4-week-old seedlings was extracted with the RNAprep Pure Plant Kit (432; TIANGEN, Sichuan, China) and submitted into ribonucleic acid sequencing using the Illumina HiSeq × Ten system with the paired-end method by Annoroad Gene Technology (Beijing) Co. Ltd. For transcriptome analysis, reference genome and annotation files with a gene model were downloaded from the SOL genomics network (SGN, http://www.sgn.cornell.edu) database directly. Raw reads were trimmed for low quality and length by Fastp v0.20.0 (Chen et al., 2018 (link)). Clean reads were aligned to the reference genome using Hisat v2.0.5 (Pertea et al., 2016 (link)) with the splice-aware method. The gene expression levels from RNA-seq were quantified by feature counts (Liao et al., 2014 (link)) and normalized by fragments per kb per million reads (FPKM) values. Differential expression analysis of two conditions/groups (two biological replicates per condition) was performed using the DESeq2 R package (1.16.1) (Love et al., 2014 (link)). The p-values were adjusted using Benjamini and Hochberg's approach for controlling the false discovery rate. Genes with an adjusted p < 0.05 found by DESeq2 were assigned as differentially expressed. The raw data were deposited in the NCBI SRA database (Accession: PRJNA605912).

To identify VCS TF genes, RNA-seq was performed with total RNA isolated from developing cambium, differentiating xylem and developing phloem cells collected from P. trichocarpa stems by LCM. A total of nine RNA-seq libraries for three biological replicates were generated using a TruSeq RNA Library Prep Kit (Illumina, RS-122-9001DOC), followed by sequencing with the Illumina HiSeq 4000 platform to obtain paired-end reads with a length of 150 base pairs (bp). To detect gene expression in OE-PtrVCS2 transgenic lines, cambium cell mixture was collected by scraping slightly on the inner side of bark peeled from the WT and transgenic P. trichocarpa stems using a double-edged razor blade. A total of six RNA-seq libraries for three biological replicates were generated using a NEBNext Ultra RNA Library Prep Kit (NEB, 7530), followed by sequencing with the Illumina HiSeq × Ten system to obtain paired-end reads with a length of 150 bp. After the sequencing data were filtered with SOAPnuke^{67 (link)}, the clean reads were aligned to the P. trichocarpa genome v.3.0 (Phytozome) by using Bowtie2 (ref. 68 (link)). The raw counts were determined and normalized following our established analysis pipeline^{42 (link)}. DEGs were characterized by FDR < 0.05 by using DESeq2 (ref. 69 (link)).