Beads adsorption method. The first and second-strand cDNAs were synthesized using Oligo (dT) primers and

Beads adsorption method. The first and second-strand cDNAs were synthesized using Oligo (dT) primers and digested with restriction enzyme NlaIII, which recognizes the CATG sites. The 3′ cDNA fragments were purified and the Illumina adaptor 1 was ligated to the 5′ end of the fragments through CATG sticky site. The junction of Illumina adaptor 1 and CATG site is the recognition site of MmeI, which is a type of endonuclease with separated recognition sites and digestion sites. It cuts at 17 bp downstream of the CATG site, producing tags with adaptor 1. After removing 3′ fragments with magnetic beads precipitation, Illumina adaptor 2 was ligated to the 3′ ends of the tags, thus generating a tag library with different adaptors at both ends of the tags. After 15 cycles of GDC-0084 web linear PCR amplification, 105 bp fragments were purified by 6 TBE PAGE gel electrophoresis. After denaturation, the singlechain molecules were fixed onto the Illumina Sequencing Chip (flowcell). Each molecule turned into a singlemolecule cluster sequencing template through in situ amplification. Then four types of nucleotides labeled by four different colors were added in, and sequencing was performed with the method of sequencing by synthesis (SBS).Ma et al. BMC Genomics (2016) 17:Page 15 ofData analysisQuantitative real-time PCR analysisRaw image data obtained from sequencing was transformed by base calling into sequence data, also called raw data or raw reads. Of the raw data, empty tags (no tag sequence between the adaptors), adaptors, low quality tags (tags containing unknown nucleotides “N”), abnormal tags (too long or too short tags), and single copy tags were removed to obtain clean tags (21 bp). To identity the gene expression patterns in populus roots, all clean tags were annotated by mapping to the sequenced genome of populus trichocarpa which covered all possible CATG + 17-nt tag sequences, allowing only 1 bp mismatch. The clean tags mapped to multiple reference sequences were filtered and the remaining clean tags were designated as unambiguous tags. For gene expression analysis, the number of unambiguous clean tags for each gene was calculated and then normalized to transcripts per million clean tags (TPM) [56, 57].Analysis and screening of DEGsThe quantitative real-time PCR was set up using SYBR Premix Ex Taq II Kit (TaKaRa) in a volume of 20 l. The reactions were performed in triplicate for each run and three biological replicates were included. The conditions for the PCR reactions were as follows: 95 for 3 m, followed by 44 PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28151467 amplification cycles at 95 for 30 s, 55 for 30 s, and 72 for 30 s. The specific primers used for the selected genes were listed in Additional file 7. For each gene, the pair of primers was designed on different exons using online program Primer 3 (http://bioinfo.ut.ee/ primer3/). The Ct values obtained for all the genes were normalized to that of the internal control 18S RNA. For the gene expression analysis, the transcript amount of these genes was determined using 2-Ct calculations. The transcript level of each gene without TSA treatment (0 M) was indicated as 1. Transcript levels (n-fold) of the examined genes under TSA treatment conditions were obtained by comparison with their transcript levels in the control sample (0 M).Diaminobenzidine (DAB) stain for hydrogen peroxideBased on the method described by Audic and Claverie [58], a rigorous algorithm was used to identify differentially expressed genes (DEGs) between two sample.