Truseq mrna library prep kit

Infiltrated leaf tissue was harvested 3 days post infiltration. Total RNA was extracted from the infiltrated leaf tissue using RNeasy Plant Mini Kit (Qiagen). RNAseq libraries were prepared from the triplicate samples for each type of infiltration, using 500 ng of total RNA, and indexed with the TruSeq mRNA Library Prep Kit according to the protocol (Illumina). The same amount of each RNAseq library was pooled and run on one lane of HiSeq2000 100PE (Illumina).
The quality of the sequencing reads was assessed with FASTQC [60 ]. RNA sequencing reads were trimmed using DynamicTrim (Phred score ≥20) and filtered on length (≥ 25 bp) using LengthSort [63 (link)]. RNAseq reads were aligned against the N. benthamiana transcriptome [64 (link)] using Bowtie2 v2.1.0 [65 (link)], and RSEM v1.2.3 [66 ] generated raw read counts for each transcript. DESeq [67 (link)] was used to run three differential expression analysis tests: (1) between Buffer infiltrated and Agrobacterium infiltrated; (2) between Agrobacterium and Agrobacterium containing AcMYB10; (3) between Agrobacterium and Agrobacterium containing AtLEC2. Lists of differentially expressed transcripts with a FDR adjusted P value <0.001 are shown in Additional file 7: Table S5, Additional file 8: Table S6, and Additional file 9: Table S7, respectively.

Preliminary characterization of the isolates was performed by Sanger sequencing. Amplicons were cleaned using DNA Clean & Concentrator Kit (ZymoResearch, US). Chromatogram files were prepared and edited to generate consensus sequences using BioEdit v7.2.5 [15 ].
A subset of the samples was also prepared for whole genome sequencing. RNA extracts were used as input to perform library preparation by using Truseq mRNA Library Prep kit (Illumina, San Diego, CA, USA), following the manufacturer’s recommended protocol which was modified to exclude the mRNA clean-up steps [5 (link)]. The prepared libraries were sequenced on an Illumina Miseq platform (Illumina, San Diego, CA, USA) using a 2 × 300 base paired-end reads. Sequence analysis was performed by making use of NGS Mapper v1.5 pipeline (ngs_mapper). The pipeline performs a number of sequential steps on the raw sequence reads, which includes; adapter trimming, raw sequence read cleanup, reference-based mapping and assembly and finally consensus sequence generation. The sequences reported in this study are available in GenBank under accession numbers MW314021-MW314035.

RKO, A549, and H460 cells were plated and treated for 24 h as described above, followed by harvesting in ice-cold PBS. Total RNA was extracted from cell pellets using TRI Reagent (Sigma-Aldrich), according to the manufacturer’s instructions. RNA quality was assessed using Bioanalyzer RNA 6000 Pico chips (Agilent). Poly-A( + ) RNA enrichment and strand-specific library preparation were carried out using a TruSEQ mRNA library prep kit (Illumina). Single-end 150 bp sequencing was carried out on the Illumina HiSeq 4000 platform by the Genomics Core facility at the University of Colorado Anschutz.

Total RNA was isolated from 200 endosperms dissected 40 h after seeds incubation at 30 °C and from 200 embryos dissected 4 h after seeds imbibition and growing for 40 h at 30 °C. Total RNA was isolated as previously described^{51 (link)}. cDNA libraries were prepared from 200 ng of total RNA using a TruSeq mRNA Library Prep Kit (Illumina). cDNA libraries were normalized and pooled then sequenced using HiSeq 2500 (Illumina) with single-end 50 bp reads. Transcript assembly and normalization was performed with the Cufflinks program and gene expression levels were calculated in FPKM (Fragments Per Kilobase of exon per Milion mapped fragments) units. Differential gene expression analysis was performed by Cuffdiff, a part of the Cufflinks package^{54 –56 (link)}.
GO enrichment analysis for biological processes was performed using the TAIR publicly available tool (https://www.arabidopsis.org/tools/go_term_enrichment.jsp). The differentially expressed genes (DEGs) with log₂ fold change ≥1 or ≤1and P ≤ 0.05 were selected for GO enrichment analysis. The list of genes bound by PIF3 was previously described Zhang et al.^{32 (link)}. Gene lists for abscisic acid metabolic process, abscisic acid-activated signaling pathway, gibberellin metabolic process, and gibberellin mediated signaling pathway were obtained from TAIR. Heat maps were generated using the heatmap.2 function of the gplots package in R.

Transcriptome sequencing was performed in triplicate in NK92-MI cells after incubation with exosomes (50 µg/mL) loaded with blank vector (termed Vector exo 1, 2, and 3) or overexpressed lncRNA vector (termed oe-lnc-SHNG10 exo 1, 2, and 3) for 24 h. The Illumina TruSeq mRNA Library Prep Kit was used to construct RNA sequencing libraries according to the manufacturer’s instructions. Yingbio Technology was then commissioned to perform RNA sequencing on an Illumina HiSeq 2500 platform with a paired-end 150-bp read run. The results of the quality control of raw reads, differentially expressed gene (DEG) analysis, and GO and KEGG analyses were consistent with the RNA sequencing described above. The sequencing coverage and quality statistics for each sample are summarized in Additional file 5: Table S3.