Nebnext adaptor

For library construction, mRNA was purified from 3 μg of total RNA of each sample using poly-T oligo-attached magnetic beads (New England Biolabs) [27 (link)]. Transcriptome sequencing libraries were generated using an Illumina NEBNext® UltraTM RNA Library Prep Kit (New England Biolabs) according to the manufacturer’s instructions and index codes were added to attribute the sequences to the corresponding sample. Briefly, purified mRNA was cut into fragments and the cleaved mRNA fragments were reverse-transcribed into first-strand cDNA using random hexamers, followed by synthesis of double-strand cDNA. After blunting ends, the 250 to 300 bp fragments were purified using the AMPure XP system (Beckman Coulter, Brea, CA, USA). The purified cDNA fragments were then linked using an NEBNext Adaptor (New England Biolabs) with a hairpin loop structure and amplified by PCR. The AMPure XP system (Beckman Coulter) was used to purify the PCR products and the sample library quality was assessed using an Agilent Bioanalyzer 2100 system.

We used approximately 5 ug of total RNA for each sample and used the Ribo-Zero™ rRNA Removal Kit (Illumina, San Diego, CA) to eliminate ribosomal RNA interference according to manufacturer’s instructions. The residue was purified by two rounds of ethanol precipitation, after which divalent cations were used to splice the remaining RNAs into small fragments under high temperature. The small RNA fragments were reverse-transcribed to complementary DNA (cDNA) and second strand cDNA was synthesized using Escherichia coli DNA polymerase I, deoxyuridine triphosphates (dUTPs), and RNase H. After adding an A-base to the 3ʹ ends of each cDNA fragment and ligating to the NEBNext Adaptor [New England Biolabs (NEB), Ipswich, MA], we purified the library using the AMPureXP system (Beckman Coulter, Brea, CA) and prepared for hybridization. Next, approximately 2 µl USER enzyme (NEB) was added to the cDNA buffer for PCR. TruSeq SR Cluster Kit v3-cBot-HS (Illumia) was used to cluster the samples on the cBot Cluster Generation System. Finally, the Illumina HiSeq 2500/2000 sequencing platform was used to sequence the libraries (150 bp paired-end reads) after cluster generation. Sequencing data were analyzed against the reference genome, after which the circRNAs were filtered out. The software Find_circ and CIRI2 (Zeng et al., 2017 (link)) were used to identify circRNAs;

Genomic DNA was fragmented using NEBNext dsDNA Fragmentase (NEB, Ipswich, MA, USA) following by DNA ends repairing. End-repaired DNA fragments were dA-tailed and ligated with the NEBNext adaptor (NEB, Ipswich, MA, USA). Biotinylated RNA library baits and magnetic beads were mixed with the barcoded library for targeted regions selection using the SureSelect Human All Exon V6 Kit (AgilentTechnologies, Palo Alto, Calif.). The captured sequences were further amplified for 150 bp paired-end sequencing in Illumina X-ten system (Gene Denovo Biotechnology Co. China). To identify somatic SNV, the Burrows-Wheeler Aligner (BWA) was used to align the clean reads from each sample against the human reference genome (GRCh38). Somatic CNV was identified using VarScan 2 with the following parameter: phred base quality ≥ 20, minimum coverage ≥ 20. Mutational signatures were deciphered by using a non-negative matrix factorization method.

RNA-seq libraries were generated using the NEB Next Ultra Directional RNA Library Prep Kit for Illumina (NEB). Poly-A-tailed mRNA was isolated from total RNA using oligo-dT beads. Purified mRNA was then fragmented with heat in fragmentation buffer. First strand and second strand cDNA syntheses were performed in accordance with the manufacturer’s recommendations. Second strand cDNA was then end-repaired, ligated to an NEBNext Adaptor and individually indexed, followed by limited-cycle (10) amplification. Indexed libraries were pooled and sequenced on an Illumina HiSeq 2500. FASTQ files were generated by CASAVA (v1.8.2). Galaxy workflow for RNA-Seq (www.usegalaxy.org) was used for subsequent data analysis. Reads were mapped to the human reference genome (GRCh38) using Bowtie2 (Galaxy Version 2.3.4.1). The gene expression values (Fragments Per Kilobase Million (FPKM)) were calculated by Cuffdiff (Galaxy Version 2.2.1.5) using the human NONCODEv5 transcript reference. The complete RNA-seq data along with processing protocols have been deposited in NCBI’s Gene Expression Omnibus and is accessible through GEO series accession number GSE115139.

Wild P. cablin plants were collected from Fenglai village, Yangchun City, Guangdong Province, China. Total chloroplast DNA was extracted from fresh leaves using Tiagen Plant Genomic DNA Kit (Beijing, China). Genomic DNA was fragmented into 300-bp using Covaris M220 Focused-ultrasonicator (Covaris, Woburn, MA, USA). Library preparation was conducted using NEBNext^® Ultra™ DNA Library Prep Kit Illumina (New England, Biolabs, Ipswich, MA, USA). Briefly, fragments were end-repaired (End Repair Reaction Buffer (10×) and End Prep Enzyme Mix), ligated with adaptors (NEBNext Adaptor and Blunt/TA Ligase Master Mix), removed uracil nucleotides from adapters, and purified (AMPure XP Beads, New England, Biolabs, Ipswich, MA, USA). The ligated DNA (with a 300-bp insertion) was then amplified by 6 cycles of PCR (Universal PCR Primer and Index (X) Primer; pre-denature 98 °C for 10 s, denature 98 °C 10 s, annealing 60 °C 30 s, elongation 72 °C 30 s). Finally, the PCR products were purified using AMPure XP Beads.