Peasy t1 cloning kit

An OminiPlant RNA Kit (Kangweishiji, Beijing, China) was used to extract RNA from new leaves, old leaves, root and shoot tips of hydroponic seedlings. TransScript^® First-Strand cDNA Synthesis SuperMix (TransGen Biotech, Beijing, China) was used to synthesize the cDNA First Strands. RNA and cDNA were assessed by 1.0% agarose gel electrophoresis. The whole sequence of MbNAC25 was obtained by polymerase chain reaction (PCR) with primers MbNAC25F and MbNAC25R, using the first-strand cDNA of M. baccata as a template. The primers used in this study are shown in Table 1. The obtained DNA fragments were purified and cloned into objective vectors using the pEASY^®-T1 Cloning Kit (TransGen Biotech, Beijing, China) and sequenced [36 (link),37 (link),38 (link),39 (link)].

The OminiPlant RNA Kit (Kangweishiji, Beijing, China) was used for RNA extraction. Then, RNA was used as the template to synthesize the first-strand cDNA with TransScript^® First-Strand cDNA Synthesis SuperMix (TransGen Biotech, Beijing, China) [53 (link)]. RNA and cDNA were assessed by 1.0% agarose gel electrophoresis [54 ]. The CDS region of MdMYB25 (NM_001293983.1, M. domestica) was used as the reference sequence, and primers (MbMYB4-F and MbMYB4-R, Supplementary Table S1) were designed with Primer 5.0 software. After the primers were synthesized, they were used for the amplification of MbMYB4. The purified DNA was linked to the pEASY^®-T1 Cloning Kit (TransGen Biotech, Beijing, China) and sequenced [55 (link),56 ].

The full-length cDNA of the putative NbFIE genes were amplified using the primers NbFIEfF/NbFIEfR. One microlitre of 20 μl first-strand cDNA reaction product was used as a template for PCR. The PCR products were gel purified and cloned with a pEASY-T1 cloning kit (TransGen Biotech, Beijing, China), and then sequenced to verify their identity as two putative homologues of the FIE gene. The sequence alignment was done using Clustal W (http://srs.ebi.ac.uk/) while the phylogenetic tree was constructed using Bioedit software. To clone the NbGH3.6 full-length cDNA, the 3′-RACE and 5′-RACE reaction was performed with the GeneRacer RACE Ready cDNA kit (Invitrogen, USA). The cDNA from the NbFIE-silenced plants acted as the PCR template and the NbGH3.6 gene-specific primers are listed in Supplementary Table S3 at JXB online.

In the validation cohort, 72 pairs of tumor and adjacent normal pancreatic tissues were collected from PDAC patients, and the CG sites of the promoter regions of four potential PMDGs were examined using bisulfite sequencing polymerase chain reaction (BSP). First, the upstream 3-kb DNA sequence of the gene promoter region was extracted from the NCBI dataset (Supplementary Figure S1). We predicted the CpG island with MethPrimer and selected the CpG island closest to the location of the probe in methylation microarrays (Supplementary Figure S2) to perform the subsequent cloning sequencing. The primer was designed with Primer5 software V5.6 (Table 1) according to the sequence of the CpG island. After that, the Universal DNA Purification Kit (DP214; Tiangen, Beijing, China) was used to extract and purify DNA from tissue samples following the manufacturer’s manual. The EZ DNA Methylation-Direct Kit (D5020; Zymo Research, CA, U.S.A.) and pEASY®-T1 Cloning Kit (CT101-01; TransGen Biotech, Beijing, China) were used to perform the DNA bisulfite conversion and polymerase chain reaction (PCR) cloning sequencing. We used the BiQ Analyzer to analyze the original sequencing data and performed a comparison of methylated CG sites (Supplementary Figure S3).

The degenerate primers used for amplification of cellulase and hemicellulase genes are listed in Table 1, they are according to the method described by Wang et al. [22 (link)]. The purified bacterial genomic DNA was used as a template and underwent a touchdown PCR. The PCR was performed using a thermal cycler in a 50 μL reaction mix. The detail amplification procedures for the targeted gene families are shown in Table 2. The PCR-amplified gene products were cloned using the TA cloning method of the pEASY-T1 cloning kit (Transgen, Beijing, China) according to the manufacturer’s instructions for sequencing. Clones from each enzyme family were selected for sequencing, which was conducted by Sangon Biotech (Shanghai) Co., Ltd.