Col-0 and myb112-4 mutant seedlings were grown under continuous white light (23.61 μmol m -2 s -1 ) conditions for 4 d before sampling. Total RNA was extracted using an E.Z.N.A. Plant RNA Kit (Omega Bio-tek). RNA-seq libraries were prepared, and their qualities were determined using an Agilent 2100 Bioanalyzer from Novogene Bioinformatics Technology Co. Ltd. and then sequenced using the Illumina method. The raw reads were cleaned and mapped to the Arabidopsis TAIR10 genome. Differential gene expression analysis was performed using the edgeR likelihood ratio test (Robinson et al. 2009) . Genes with false discovery rate (FDR) < 0.05 were selected for subsequent analysis.
E z n a plant rna kit
Manufactured by Omega Bio-Tek
Sourced in United States, China
The E.Z.N.A. Plant RNA Kit is a laboratory equipment product designed for the isolation and purification of high-quality RNA from plant tissues. It utilizes a silica-based membrane technology to efficiently capture and purify RNA, allowing for its subsequent analysis and research applications.
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Lab products found in correlation
Primescript rt reagent kit, by Takara Bio (8 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (7 mentions)
Primescript rt reagent kit with gdna eraser, by Takara Bio (6 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (5 mentions)
Sybr green, by Takara Bio (5 mentions)
Primescript rtase, by Takara Bio (5 mentions)
Dnase 1, by Thermo Fisher Scientific (4 mentions)
Rq1 rnase free dnase, by Promega (4 mentions)
Hiscript 2 1st strand cdna synthesis kit, by Vazyme (4 mentions)
Chamq universal sybr qpcr master mix, by Vazyme (4 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (4 mentions)
Itaq universal sybr green supermix, by Bio-Rad (4 mentions)
Nanodrop 1000, by Thermo Fisher Scientific (4 mentions)
Iscript cdna synthesis kit, by Bio-Rad (4 mentions)
Hiseq 2500, by Illumina (4 mentions)
Hiseq 4000, by Illumina (3 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (3 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (3 mentions)
Abi7500 real time pcr amplifier, by Thermo Fisher Scientific (3 mentions)
Agilent bioanalyzer 2100 system, by Agilent Technologies (3 mentions)
159 protocols using e z n a plant rna kit
1
Differential Gene Expression in Arabidopsis
2
Arabidopsis RNA Extraction and RT-qPCR
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Total RNA was isolated from Arabidopsis seedlings using an E.Z.N.A. Plant RNA Kit (Omega Bio-tek). For reverse transcription, 1 μg of total RNA was used as a template to generate first-strand cDNA using the All-In-One 5× RT Master Mix (Applied Biological Materials). RT-qPCR was performed using the ABI QuantStudio™ 6 Flex Real-Time PCR System following the manufacturer's instruction. The relative expression level of genes was analyzed using the 2 -ΔΔCt method, with the value of PP2A expression serving as an internal control.
3
Quantitative PCR Analysis of Transgenic Arabidopsis
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Total A. thaliana RNA was extracted from entire WT and twelve transgene line (SBP16-1, SBP16-8, SBP16-14, SBP16-18, SBP16-23, SBP16-32, SBP16-35, SBP16-40, SBP16-47, SBP16-57, SBP16-61 and SBP16-67) plants using the E.Z.N.A.® Plant RNA Kit (Omega Bio-tek, Norcross, GA, USA). Cycling parameters were as follows: 94 °C for 3 min, 25 cycles at 94 °C for 30 s, 60 °C for 30 s and 72 °C for 30 s, with a final elongation step at 72 °C for 10 min. An 8 μL sample of each PCR product was subsequently separated on a 1.2% (w/v) agarose gel, then stained with ethidium bromide and photographed under UV light. Atactin1 (At2g37620) was amplified for use as an internal control. VpSBP16-F, VpSBP16-R and AtActin1-F, AtActin1-R specific primer sequences are listed in Table 1 .
4
Quantifying GUS Transcript Levels in UV-C Treated Tobacco
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GUS transcript levels in UV-C treated or untreated tobacco leaves were quantified by two-step qRT-PCR. Total RNA was isolated from tobacco leaf samples using the E.Z.N.A. ® Plant RNA Kit (Omega Bio-tek, USA, R6827-01), and the RNA concentration was adjusted to 500 ng for reverse transcription. The reactions were carried out in triplicate in a final volume of 25 μl using an iCycler iQ5 thermal cycler (Bio-Rad, Hercules, CA, USA), and a SYBR Premix ExTM TaqII kit (Takara). Cycling parameters were 95°C for 30 s, followed by 40 cycles of 95°C for 5 s and 62°C for 30 s. The tobacco ubiquitin gene (UBI, GenBank Accession number U66264 ) was used as the internal control. GUS transcript levels were calculated using the normalized-expression method. The following primers were used for real-time PCR amplifications: 5′ - ATTATGCGGGCAA CGTCTGGTATCAG - 3′ and 5′ - CAT CGGCTTCA AATGGCGTAGC - 3′ for GUS, 5′ - ATGAACGC TGGCGGCATG CTTA - 3′ and 5′ - AGATCTGCATTC CTCCCCTCGCTA - 3′ for UBI.
5
Quantitative Real-Time PCR Analysis of Gene Expression
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Total RNA was extracted from the leaves of each cultivar under each condition using an E.Z.N.A. plant RNA kit (OMEGA Bio-Tek, USA), and three biological replicates were used for qRT-PCR analysis. Firststrand cDNA was synthesized by using Superscript III reverse transcriptase (Invitrogen). qRT-PCR was performed on an ABI Prism 7500 real-time PCR system (Applied Biosystems, USA). For qRT-PCR analysis, each lncRNA-and mRNA-speci c primer was designed by Primer3 (Table S1). The expression level of the rice Actin1 gene (accession no. X16280) was used as the internal control with primers forward 5′-TGGCATCTCTCAGCACATTCC-3′ and reverse 5′-TGCACAATGGATGGGTCAGA-3′. q-PCR ampli cations were performed in an optical 384-well plate. Each reaction was performed in a volume of 25 µL containing 12.5 μL of 2× SYBR green master reagent (Applied Biosystems, COUNTRY NAME), 5.0 μL diluted transcription product, 0.2 μL of each gene-speci c primer and 7.1 µL ddH 2 O. The following thermal cycle was used: 95 °C for 3 min and then 45 cycles of 95 °C for 30 s, 60 °C for 30 s and 72 °C for 1 min. Dissociation curve analysis was performed using the following thermal pro le: 95 °C for 15 s, 60 °C for 20 s and 95 °C for 15 min. The expression levels relative to actin gene were normalized as 2 - △△CT (Livak & Schmittgen 2001).
6
RNA Extraction and Sequencing Protocol
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RNA for the expression atlas was extracted using the Omega Biotek E.Z.N.A.® Plant RNA Kit according to the manufacturer’s protocol. Roughly 200 mg of ground tissue was used for each extraction. The RNA quality was validated using gel electrophoresis and the Qubit RNA IQ Assay (Thermo Fisher). Stranded RNAseq libraries were constructed using 2 μg of total RNA quantified using the Qubit RNA HS Assay Kit (Invitrogen, USA) with the Illumina TruSeq stranded total RNA LT Sample Prep Kit (RS-122-2401 and RS-122-2402). Multiplexed libraries were pooled and sequenced on an Illumina HiSeq4000 under paired-end 150 nt mode. Three replicates were sequenced for each timepoint/sample.
7
Quantification of gene expression via RT-qPCR
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Total RNA from ground whole leaf blades was extracted using the E.Z.N.A Plant RNA Kit (Omega Bio-Tek, VWR) or TRIzol reagent. DNase treatment of total RNA was performed by DNaseI (Invitrogen, Thermo-Fischer) followed by reverse transcription using 1 μg RNA with the RevertAid First Strand cDNA Synthesis Kit (Thermo Fischer Scientific, USA) or MMLV reverse transcriptase (Promega), according to the manufacturer’s instructions. Transcript levels were analyzed via reverse transcription quantitative PCR (RT-qPCR), using LightCycler480 SYBR Green I Master mix (Roche) and the LightCycler 480. Data were analyzed using LightCycler 480 Software Release1.5.0. Relative gene expressions was calculated according to Pfaffl (2001) (link) or the 2−ΔΔCT method. Primer efficiency was determined using a standard curve of a mixture of cDNAs. TIP41 and Hrd1a (Czechowski et al., 2005 (link)), or ACT2 were used as reference genes. Primers are listed in Supplemental Table S1 . For each time point, at least three biological replicates were analyzed. Statistical difference was assessed using Student’s t test.
8
Quantification of Gene Expression in Endometrial Tissues
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RNA was extracted from 0.05 g of each sample (EC and NEC tissues) using an E.Z.N.A. Plant RNA Kit (Omega Bio-tek Inc., Norcross, GA, USA) following the manufacturer’s instructions. Genomic DNA was removed and first strand cDNA was synthesized using the RT Reagent Kit (Takara, Dalian, China) following the manufacturer’s instructions. The cDNA was stored at -80°C until use. All samples were analyzed in triplicate.
Ubiquitin-conjugating enzyme (UBC), a widely used reference gene whose expression was not significant in EC and NEC, was selected for real-time PCR normalization (Niu et al., 2015 (link)). The primers for genes are shown inTable S1
Ubiquitin-conjugating enzyme (UBC), a widely used reference gene whose expression was not significant in EC and NEC, was selected for real-time PCR normalization (Niu et al., 2015 (link)). The primers for genes are shown in
9
Tomato Leaf RNA Extraction and qRT-PCR
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Total RNA was extracted from tomato leaves using an E.Z.N.A.® Plant RNA Kit (Omega Bio–Tek, Doraville, GA, USA) according to the manufacturer’s instructions. The total RNA was then reverse–transcribed using a PrimeScriptTM RT reagent kit with gDNA Eraser (Takara, Shiga, Japan) in a 20–μL reaction mixture containing 1 μL of total RNA from each individual sample. Real–time PCR was performed on a CFX96™ real–time PCR cycler (Bio–Rad, Hercules, CA, USA) and a SYBR Premix Ex Taq (TliRNaseH Plus) Kit (Takara). Initial denaturation at 95 °C for 30 s was followed by 40 cycles of 95 °C for 5 s, 58 °C for 30 s, and a melting curve of 65–95 °C. Primers for the actin gene were used as an internal control. Primers for psbA and actin were designed as described by Wu et al. [55 (link)]. Primers for the pbgD and Chlase genes were designed using Primer3, version 4.0.0 (website software), with the primer length set at 20 − 24 bp; melting temperature of 58 − 62 °C; CG content, 30 − 70 %; and product size, 150–250 bp. All samples were analyzed three times.
10
Gene Expression Analysis in P. cathayana
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The total RNA of leaves and roots was extracted using the E.Z.N.A.® Plant RNA Kit (Omega Bio-Tek, Norcross, GA, United States). The synthesis of first-strand cDNA was carried out with 1 μg RNA by using the reverse transcription kit (HiScript® II Reverse Transcriptase, Vazyme). Then the cDNA was diluted 5 times for quantitative real-time PCR (qRT-PCR) analysis. PcGLL and Pctublin were the internal reference genes of P. cathayana qRT-PCR. The primers for qRT-PCR are shown in Supplementary Table 1 . The reaction system was 10 μL: 5 μL 2 × ChamQ SYBR qPCR Master Mix (Vazyme), 1 μL forward primer, 1 μL reverse primer, 1.5 μL DEPC water, and 1.5 μL cDNA. The reaction conditions were: 95°C for 3 min; 39 cycles at 95°C for 10 s, 58°C for 20 s, and 72°C for 20 s; followed by 95°C for 10 s, and then 61 cycles of 5 s increasing from 65°C to 95°C by an increment of 0.5°C for each cycle. The 2–ΔΔCt method (Livak and Schmittgen, 2001 (link)) was used to calculate the relative quantitative values of different genes.
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