Quantseq fwd kit

Gene expression profiling was conducted using a 3’-Tag-RNA-Seq protocol. Barcoded sequencing libraries were prepared using the QuantSeq FWD kit (Lexogen, Vienna, Austria) for multiplexed sequencing according to the recommendations of the manufacturer using both the UDI-adapter and UMI Second-Strand Synthesis modules (Lexogen). The fragment size distribution of the libraries was verified via micro-capillary gel electrophoresis on a LabChip GX system (PerkinElmer, Waltham, MA). The libraries were quantified by fluorometry on a Qubit fluorometer (LifeTechnologies, Carlsbad, CA), and pooled in equimolar ratios. The library pool was quantified via qPCR with a Kapa Library Quant kit (Kapa Biosystems/Roche, Basel, Switzerland) on a QuantStudio 5 system (Applied Biosystems, Foster City, CA). Up to 48 libraries were sequenced per lane on a HiSeq 4000 sequencer (Illumina, San Diego, CA) with single-end 100 bp reads at UC Davis Genome Center.

RNA sequencing (RNA-seq) libraries were prepared from the RNA of cortex sections (Table 1). Sequencing and library preparation were performed by the DNA technologies and Expression Analysis Core in the Genome Center of the University of California, Davis. RNA Integrity (RIN) scores were assessed for all samples, resulting in a mean of 6.4 ± 0.87 (range = 4.9–8.3). Gene expression profiling was carried out using a 3′-Tag-RNA-Seq protocol. Barcoded sequencing libraries were prepared using the QuantSeq FWD kit (Lexogen, Vienna, Austria) for multiplexed sequencing according to the recommendations of the manufacturer starting from 300 ng total RNA each. Both the unique dual index (UDI)-adapter and unique molecular identifier (UMI) Second Strand Synthesis modules were used (Lexogen). The fragment size distribution of the libraries was verified via microcapillary gel electrophoresis on a LabChip GX system (PerkinElmer, Waltham, MA). The libraries were quantified by fluorometry on a Qubit fluorometer (Life Technologies, Carlsbad, CA) and pooled in equimolar ratios. The library pool was quantified via quantitative PCR (qPCR) with a KAPA Library Quantification Kit (Kapa Biosystems/Roche, Basel, Switzerland) on a QuantStudio 5 system (Applied Biosystems, Foster City, CA). The libraries were sequenced on a HiSeq 4000 sequencer (Illumina, San Diego, CA) with single-end 100-bp reads.

Total RNA was extracted with TRIzol (Fisher #15596018), following the manufacturer’s instructions, for intact infiltrated leaves as well as leaf protoplasts isolated using the above mentioned method for scRNA-seq. DNase treatments were performed with RQ1 RNase-Free DNase (Promega #PR-M6101). Three biological replicates were performed for samples of Pst DC3000- or mock-infiltrated leaves, and one repeat was made for leaf protoplasts of each sample. cDNA libraries were prepared with QuantSeq FWD kit (Lexogen), according to the manufacturer’s protocol. The fragment size distribution was evaluated by a Bioanalyzer 2100 (Agilent). The library pool was treated using Exonuclease VII (NEB), SPRI-bead purified with KapaPure beads (Kapa Biosystems /Roche), quantified via qPCR with a Kapa Library Quant kit (Kapa Biosystems) on a QuantStudio 5 RT-PCR system (Applied Biosystems). Sequencing was performed at the University of California Davis Genome Center using a HiSeq 4000 (Illumina) platform with single-end 100 bp reads.