Raw RNA‐seq data used in this work are available from the NCBI Gene Expression Omnibus under accession numbers GSE106811 , GSE116194 , and GSE138039 . Samples and RNAs were prepared as described in Sucher et al. (2020 ). Briefly, the edge of 25 mm‐wide developed necrotic lesions were isolated with a scalpel blade and immediately frozen in liquid nitrogen. Samples were harvested before lesions reached 25 mm width, at 24 hr (H. annuus), 47–50 hr (A. thaliana, P. vulgaris, R. communis, and S. lycopersicum) or 72 hr postinoculation (B. vulgaris). Material obtained from leaves of three plants were pooled together for each sample, all samples were collected in triplicates. RNA extractions were performed using NucleoSpin RNA extraction kits (Macherey‐Nagel) following the manufacturer's instructions. RNA sequencing was outsourced to Fasteris SA to produce Illumina single‐end reads (A. thaliana, S. lycopersicum, in vitro control) or paired reads (other infected plants) using a HiSeq 2500 instrument.
Nucleospin rna extraction kit
Manufactured by Macherey-Nagel
Sourced in Germany, United States, France
The NucleoSpin RNA extraction kit is a product designed for the purification of total RNA from various biological samples. It utilizes a silica membrane technology to efficiently capture and purify RNA molecules.
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Lab products found in correlation
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (17 mentions)
Iscript cdna synthesis kit, by Bio-Rad (9 mentions)
Maxima first strand cdna synthesis kit, by Thermo Fisher Scientific (8 mentions)
Nanodrop 1000 spectrophotometer, by Thermo Fisher Scientific (8 mentions)
Nanodrop 1000, by Thermo Fisher Scientific (8 mentions)
Itaq universal sybr green master mix, by Bio-Rad (6 mentions)
Nanodrop, by Thermo Fisher Scientific (6 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (6 mentions)
Itaq universal sybr green supermix, by Bio-Rad (5 mentions)
Iscript reverse transcriptase, by Bio-Rad (5 mentions)
Lightcycler 480, by Roche (4 mentions)
Superscript 4, by Thermo Fisher Scientific (4 mentions)
Quantittect sybr green pcr kit, by Qiagen (4 mentions)
Multiscribe reverse transcriptase, by Thermo Fisher Scientific (4 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (4 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (4 mentions)
Sybr green supermix, by Bio-Rad (3 mentions)
7900ht fast real time pcr system, by Thermo Fisher Scientific (3 mentions)
M mlv reverse transcriptase, by Promega (3 mentions)
Itaq sybr green, by Bio-Rad (3 mentions)
150 protocols using nucleospin rna extraction kit
1
RNA-seq Protocol for Plant Pathogenesis
2
RNA-seq analysis of CD8+ T cells
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RNA from 0.25 to 1x106 CD8+ T cells was isolated with Nucleospin RNA extraction kits (Macherey Nagel, catalog #740955.250); libraries were prepared using an Illumina TruSeq Library Kit and sequenced with an Illumina NovaSeq instrument. Reads were aligned on reference genomes (mm10 for mouse data, GRCh38 for human data) using the STAR universal RNA-seq aligner; DEGs were calculated with DESeq2. Heatmaps were created with Morpheus (Morpheus (broadinstitute.org)). For functional enrichment analyses, we used the enricher function (default parameters) from the clusterProfiler package v4.2.2 to perform hypergeometric tests for functional enrichment analysis. Only down-regulated genes were used as the input, and the universe/background was defined as all detected genes in our RNA-Seq. The human hallmark, C2 and GOBP (C5:BP) gene sets were retrieved from the Molecular Signatures Database [MSigDB (20 (link))] using the msigdbr function and package v7.4.1 Finally, we applied the Benjamini-Hochberg method to control false discoveries in multiple hypothesis testing.
3
Comparative Transcriptomics of Plant-Pathogen Interactions
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Raw RNA-seq data used in this work is available from the NCBI Gene Expression Omnibus under accession numbers GSE106811, GSE116194 and GSE138039. Samples and RNAs were prepared as described in (Sucher et al., 2020) . Briefly, the edge of 25 mm-wide developed necrotic lesions were isolated with a scalpel blade and immediately frozen in liquid nitrogen. Samples were harvested before lesions reached 25 mm width, at 24 hours (H. annuus), 47 to 50 hours (A. thaliana, P. vulgaris, R. communis and S. lycopersicum) or 72 hours post inoculation (B.
vulgaris). Material obtained from leaves of three plants were pooled together for each sample, all samples were collected in triplicates. RNA extractions were performed using NucleoSpin RNA extraction kits (Macherey-Nagel, Düren, Germany) following the manufacturer's instructions.
RNA sequencing was outsourced to Fasteris SA (Switzerland) to produce Illumina single end reads (A. thaliana, S. lycopersicum, in vitro control) or paired reads (other infected plants) using a HiSeq 2500 instrument.
vulgaris). Material obtained from leaves of three plants were pooled together for each sample, all samples were collected in triplicates. RNA extractions were performed using NucleoSpin RNA extraction kits (Macherey-Nagel, Düren, Germany) following the manufacturer's instructions.
RNA sequencing was outsourced to Fasteris SA (Switzerland) to produce Illumina single end reads (A. thaliana, S. lycopersicum, in vitro control) or paired reads (other infected plants) using a HiSeq 2500 instrument.
4
Quantitative RT-PCR for Gene Expression Analysis
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Total RNA was extracted using Nucleospin RNA extraction Kit (Macherey-Nagel, Bethlehem, PA), reverse transcribed to cDNA using iScript Reverse Transcriptase Kit (BioRad, Hercules, CA). QRT-PCR was performed using iTaq Universal SYBR Green Supermix (BioRad, Hercules, CA) and detected on a QuantStudio5 Real-Time PCR system (Applied Biosystems, Foster City, CA). Primers for target genes were listed in Supplementary Tables 4 and 5 .
5
Quantitative RT-PCR for Gene and Viral Expression
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Total RNA was extracted using the NucleoSpin RNA extraction kit (Macherey-Nagel) according to the manufacturer’s instructions. cDNA was prepared using iScript (BioRad) and subjected to the real-time PCR analysis on a CFX96 instrument (BioRad), by using (2x) SYBR Green Supermix (BioRad). Data was analyzed by using the ΔΔCt method with GAPDH as an internal control. Primers used in this study are summarized in S3 Table S3 Table
6
Gene Expression Analysis in Cells
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Total RNA was extracted using the NucleoSpin® RNA extraction kit (Macherey-Nagel, Germany), as recommended by the manufacturer. Total RNAs were reverse transcripted using the iScript cDNA Synthesis Kit (Bio-Rad, USA). Genes of interest were amplified using 5x FIREPol® Master Mix (Solis BioDyne, Estonia). Primer sequences for hTERT, VEGF and GAPDH amplification were: hTERT forward primer 5’-TGAACTTGCGGAAGACAGTGG-3’ , hTERT reverse primer 5’-ATGCGTGAAACCTGTACGCCT-3’ , VEGF forward primer 5’-GGAGGGCAGAATCATCACGAAG-3’ , VEGF reverse primer 5’-CACACAGGATGGCTTGAAGATG-3’ , GAPDH forward primer 5’-TGGGATGGACTGTGGTCATGAG-3’ , GAPDH reverse primer 5’-ACTGGCGTCTTCACCACCATGG-3’ . Reactions were initiated at 95°C for 3 min, followed by 94°C, 58°C (for hTERT), or 60°C (for VEGF and GAPDH) for 30 s, and 70°C for 30 s, 40 cycles for hTERT, or 35 cycles for VEGF and GAPDH, then followed by an elongation step at 72°C for 7 min. After amplification, PCR products were separated on 2% agarose gels and visualized by SYBR® Safe DNA Gel Stain (Invitrogen, Life Technologies, USA).
7
RNA-Seq Library Preparation and Sequencing
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RNA of each larvae (n=10 per condition) used in these experiments was extracted using NucleoSpin RNA extraction kit (Macherey-Nagel, Düren, Germany).RNA-Seq libraries were generated from 2000 ng of total RNA using the Illumina Stranded mRNA Prep mRNA Sample Preparation Kit with UDI indices (Illumina, USA) according to the manufacturer’s instructions. Surplus PCR primers were removed using AMPure XP (Beckman Coulter Life Sciences, USA). Final cDNA libraries were checked for quality and quantified using Qubit (ThermoFisher Scientific, USA) and Fragment Analyzer for size profiling (Agilent, USA), and concentration normalized using KAPA Library Quantification Kit for Illumina Platforms (Roche, USA). Sequencing was performed on an Illumina NextSeq2000 for paired-end 150 base format. Libraries were loaded in the P2 flow cell at 645 nM. The fastQ files were generated and demultiplexed using bcl2fastq v2.20 pipeline. RNA-Seq library preparation and sequencing were performed by the High Throughput Genomics Core of Biodiversity Research Center in Academia Sinica in Taipei, Taiwan (http://ngs.biodiv.tw/NGSCore/contact-location/ ).
8
RNA Extraction and qPCR Analysis
9
ITPR Expression and Knockdown in Cells
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RNA was extracted by NucleoSpin® RNA extraction kit (Macherey‐Nagel #740955) and reverse transcribed by Maxima first strand cDNA synthesis kit (Thermo Fischer). RT‐qPCR was performed using DyNAmo Flash SYBR Green qPCR Kit (Thermo Scientific) with specific primers for ITPR1, ITPR2, ITPR3, and GAPDH (Table S2 ) using Bio‐Rad CFX Maestro 1.1 software (Bio‐Rad). ITPR3 siRNA knockdown experiments were conducted as described19 using ITPR3 ON‐TARGETplus SMARTpool siRNA (Dharmacon #L‐006209‐00‐0005) and ON‐TARGETplus Non‐targeting siRNA (Dharmacon #D‐001810‐01‐05).
10
Quantitative PCR RNA Analysis Protocol
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