Bioanalyzer 2100 system

Total RNA was obtained from the flowers samples using RNAprep Pure Plant Kit (Polysaccharides&Polyphenolics-rich) (Tiangen Biotech, Beijing, China) following the manufacturer’s procedure. The RNA concentration and purity were checked by OD A260/A280 (>1.8) and A260/A230 (>1.6), and the yield and quality were accessed using Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) and RNA 6000 Nano LabChip Kit (Agilent, CA, USA), RIN>7.0. The preparation of whole transcriptome libraries and deep sequencing were performed by Beijing Ori-Gene Science and Technology CoRP., LTD (Beijing, PR China). Transcriptome libraries were constructed using NEBNext^® Ultra^™ RNA Library Prep Kit for Illumina (New England Biolabs) according to the manufacturer’s instructions. Libraries were controlled for quality and quantified using the BioAnalyzer 2100 system and qPCR (Kapa Biosystems, Woburn, MA, USA). The resulting libraries were sequenced initially on a HiSeq2000/2500 instrument that generated paired-end reads of 100/150 nucleotides.

According to the manufacturer’s instructions, we used the Ribo-Zero Magnetic Gold Kit (Illumina, San Diego, CA, USA) and the NEBNext Ultra RNA Library Prep Kit for Illumina (New England Biolabs) to construct the full transcriptome library. We used the Bioanalyzer 2100 system and qPCR (Kapa Biosystems, Woburn, MA, USA) for quality inspection of the library. We sequenced using the Illumina Hiseq2000/2500 and produced read lengths of 2 × 100/150 bp. The original data was evaluated using fastqc (http://www.bioinformatics.bbsrc.ac.uk/projects/fastqc/version0.10.1), and compared with the reference genome by the TopHat 2.0 program (http://tophat.cbcb.umd.edu/version2.0.10). After the transcripts of each sample were assembled separately, the transcripts of all samples were summarized and merged using the command of cuffmerge. Then, the ensembl transcript database was used as the annotation reference for mRNA, and the number of sequences in each transcript was standardized according to the total length and samples. The number of sequences in every 1 million pairs to every 1000 bases in exon was used as the expression amount. FDR correction was applied to p value to obtain Q value as follows: The screening criteria for differential mRNA were as follows: P value < 0.05 and Q value ≤ 0.05.

The extracted RNA was prepared for RNA sequencing and deep sequencing of intermediate-size RNAs (50 to 300 nt) with two biological replicates. For RNA-seq, the total RNA quantity and purity were analyzed using a Bioanalyzer 2100 and RNA 6000 Nano LabChip Kit (Agilent, CA, USA) with RIN number > 7.0. The preparation of whole-transcriptome libraries and deep sequencing were performed by the Annoroad Gene Technology Corporation. Libraries were controlled for quality and quantitated using the Bioanalyzer 2100 system and qPCR (Kapa Biosystems, Woburn, MA). The resulting libraries were sequenced initially on a HiSeq 2000 instrument that generated paired end reads of 150 nt. For intermediate-size RNA-seq, the library size selection was performed by gel electrophoresis with a range of 50–300 bp. Approximately 1 μg of total RNA was used to prepare the library according to the protocol of the TruSeq Small RNA Sample Prep Kits (Illumina, San Diego, USA). The libraries were subsequently sequenced on the Illumina HiSeq2500 platform at LC-BIO (Hangzhou, China) following the manufacturer’s instructions, and the full-length pair-end reads were obtained. The datasets generated during the current study are available in the SRA database of NCBI (SRP338667) [68 ].

Total RNA was extracted from four tissue types (leaf, phloem, xylem, and root) from each sample for RNA sequencing using a CTAB procedure (Porebski et al., 1997 (link)). Each sample was performed in triplicate using three individual saplings treated under the same conditions. A total of 36 samples were used for the subsequent experiments with RNA integrity number (RIN) values over 8.0 for each poplar. Whole-transcriptome libraries were constructed, and deep sequencing was performed by the Annoroad Gene Technology Corporation (Beijing, China). Whole-transcriptome libraries were constructed using NEB Next Ultra Directional RNA Library Prep Kit for Illumina (NEB, Ispawich, USA) according to the manufacturer’s instructions. Libraries were controlled for quality and quantified using the BioAnalyzer 2100 system and qPCR (Kapa Biosystems, Woburn, MA, USA). To identify antisense transcripts, a strand-specific RNA-seq strategy was adopted, and RNA-seq libraries were generated using the SOLiD™ Whole Transcriptome Analysis Kit (ABI). The resulting libraries were initially sequenced on a HiSeq 2500 instrument that generated paired-end reads of 125 nucleotides. All sequencing data have been submitted to the NCBI Sequence Read Archive (SRA accession numbers SRX3504248-SRX3504283).

Total RNA was extracted as described above. The preparation of whole transcriptome libraries and deep sequencing were performed by the Annoroad Gene Technology Corporation (Beijing, PR China). Whole transcriptome libraries were constructed using TruSeq Stranded Total RNA with Ribo-Zero Gold (Illumina, San Diego, CA, USA) according to the manufacturer’s instructions. Libraries were controlled for quality and quantified using the BioAnalyzer 2100 system and qPCR (Kapa Biosystems, Woburn, MA, USA). The resulting libraries were sequenced initially on a HiSeq 2000 instrument that generated paired-end reads of 100 nucleotides.

Total RNA was obtained from rice anthers before flowering, pistils before flowering, spikelets 5 DAP and shoots 14 DAG; these samples were used for sequencing. The preparation of whole transcriptome libraries and deep sequencing were performed by the Annoroad Gene Technology Corporation (Beijing, PR China). Whole transcriptome libraries were constructed using TruSeq Stranded Total RNA with Ribo-Zero Gold (Illumina, San Diego, CA, USA) according to the manufacturer’s instructions. Libraries were controlled for quality and quantified using the BioAnalyzer 2100 system and qPCR (Kapa Biosystems, Woburn, MA, USA). The resulting libraries were sequenced initially on a HiSeq 2000 instrument that generated paired-end reads of 100 nucleotides. The sequencing data have been submitted to the NCBI Sequence Read Archive (SRA accession number SRP047482).