Foliar tissue from Arabidopsis AtMIOX4 L21 plants and wild‐type controls was collected at developmental stage 5.0. High‐quality RNA was used to make six cDNA libraries, three from each genotype. These libraries were sequenced using the Illumina HiSeq 2500 Rapid Run flow cell with 1x50 bp paired end format. A total of 200,736,389 raw reads were obtained. The percentage of adapters in samples was up to 3.1%. The Trim‐Galore script was used to remove the reads <40 bp in all samples. After removal of adapters and low‐quality reads, 185,235,265 bp of high‐quality reads were obtained. Reads that achieved a high Phred score (>38) indicating good quality sequences were used for the assembly. To assess the sequencing quality, the reads were mapped to the A. thaliana genome (TAIR10) using TopHat. The functional gene ontology annotation of transcripts was performed using the Blast algorithm. The quality and mapping of the RNA‐Seq data obtained from Illumina sequencing were good for transcriptome analysis of the AtMIOX4 OE line.
Hiseq 2500 rapid run flow cell
Manufactured by Illumina
The HiSeq 2500 Rapid Run flow cell is a laboratory equipment designed for high-throughput DNA sequencing. It provides a rapid sequencing solution with reduced runtime compared to standard flow cells. The core function of the HiSeq 2500 Rapid Run flow cell is to enable efficient and high-speed DNA sequencing as part of the HiSeq 2500 sequencing system.
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Lab products found in correlation
Bcl2fastq v1, by Illumina (3 mentions)
Truseq stranded mrna library prep kit lt, by Illumina (2 mentions)
Bioanalyzer, by Agilent Technologies (2 mentions)
Real time analysis v1, by Illumina (2 mentions)
Real time analysis software, by Illumina (1 mentions)
Rneasy plant mini kit, by Qiagen (1 mentions)
Agilent 2100, by Agilent Technologies (1 mentions)
Truseq stranded mrna sample preparation kit, by Illumina (1 mentions)
Qubit assay, by Thermo Fisher Scientific (1 mentions)
Dna 1000 kit, by Agilent Technologies (1 mentions)
Truseq dna pcr free lt sample prep kit, by Illumina (1 mentions)
Sv total rna kit, by Promega (1 mentions)
Library qpcr assay, by Illumina (1 mentions)
Hiseq rapid sbs reagents, by Illumina (1 mentions)
Dneasy blood and tissue kit, by Qiagen (1 mentions)
Truseq nano dna library preparation kit, by Illumina (1 mentions)
Nanodrop lite spectrophotometer, by Thermo Fisher Scientific (1 mentions)
10 protocols using hiseq 2500 rapid run flow cell
1
RNA-Seq Analysis of AtMIOX4 Overexpression in Arabidopsis
2
Illumina Sequencing of RNA Libraries
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The cDNA libraries were constructed for each individual using Illumina TrueSeq Stranded mRNA Library Preparation Kits LT (Illumina, San Diego, CA), with each individual receiving a uniquely identifiable index tag. The quality of each library was evaluated and the 20 individuals were multiplexed into a single sample that was subsequently run on two lanes of an Illumina HiSeq2500 Rapid Run flow cell (v1). Sequencing was performed on paired end 2 x 150 bp format reads and bases were called using Illumina Real Time Analysis software (v1.17.21.3). Reads from each individual were identified based on their unique index tag, separated, and converted to fastq files using Illumina Bcl2fastq v1.8.4. Sequencing produced an average of 14.5 million reads per individual, with over 90% of the reads having a Q-score >30.
3
RNA Extraction and Sequencing from Plant Roots
4
RNA Sequencing Sample Preparation
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Three biological replicates of each experimental treatment were selected based on quality (RIN score 7) using a BioAnalyzer Agilent 2100. Samples were pooled on two lanes of an Illumina HiSeq 2500 Rapid Run flow cell (v1) and sequenced in a 1 × 50 bp single-end format using Rapid SBS reagents. Details regarding sequencing, read counts and data analysis are provided in Supplementary Note 1 .
5
Preparation and Sequencing of RNA-seq Libraries
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Thirty-two RNAseq libraries were prepared using TruSeq Stranded mRNA Sample Preparation Kit (Illumina) following the manufacturer’s instructions. The concentration of each amplified library was measured using Qubit assays (Life Technologies) and the size distribution was assessed on a Bioanalyzer using the DNA1000 kit (Agilent Technologies). Library concentration was normalized to 10nM and pooled for multiplex sequencing. The thirty two RNA-seq libraries were prepared in 4 pools and delivered to the Research Technology Support Facility, Michigan State University, for sequencing. After quantitation and validation by Qubit (Life Technologies) and quantitative real-time PCR (KAPA Biosystems) each pool was loaded on a single lane of an Illumina HiSeq 2500 Rapid Run flow cell (v1). Sequencing was performed using TruSeq Rapid SBS reagents (Illumina) in a 2x100bp (PE100) format.
All RNAseq data has been made available via the NCBI GEO repository [22 (link)], under the accession number GSE71431.
All RNAseq data has been made available via the NCBI GEO repository [22 (link)], under the accession number GSE71431.
6
Illumina-based RNA Sequencing Workflow
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RNA sequencing including RNA quantification and library preparation was performed at the Research Technology Support Facility (RTSF), Michigan State University, East Lansing, MI, USA. The RNA was checked for integrity before library preparation and sequencing using a Bioanalyzer (Agilent Technologies). Briefly, multiplex sequencing libraries were prepared using the Illumina TruSeq Stranded mRNA Library Prep Kit LT. After QC, all six libraries (three treatment and three controls) were combined into a single pool. This pool was loaded on one lane of an Illumina HiSeq 2500 Rapid Run flow cell (v1). Sequencing was performed in a PE100 format with Rapid SBS reagents. Base calling was done by Illumina Real Time Analysis (RTA) v1.17.21.3 and output of RTA was de-multiplexed and converted to Fastq format with Illumina Bcl2fastq v1.8.4.
7
Genomic DNA Extraction and Illumina Sequencing
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Genomic DNA of mako and hammerhead for Illumina sequencing were extracted using the Epicentre MasterPure DNA purification kit (Biosearch Technologies). The sequencing libraries were created using Illumina’s TruSeq DNA PCR-Free LT Sample Prep Kit (catalog number FC-121-3001) following the manufacturer’s recommended protocol and sequenced on both lanes of a HiSeq2500 Rapid Run flowcell, as paired-end 2 × 250 bp (mako) and 2 × 125 (hammerhead) runs.
8
Transcriptome Analysis of 3D Cardiac Model
9
Whole Genome DNA Extraction and ddRADseq
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We extracted whole genomic DNA using the DNeasy Blood and Tissue Kit (Qiagen Inc., Valencia, CA), using the standard blood and tissue protocols as appropriate. Each individual was barcoded and genotyped following a ddRADseq (Peterson et al., 2012 ) protocol using the SphI‐EcoR1 enzyme combination and isolating fragments in the range of 345–407 base pairs (bp). Details of the protocol can be found in the supplemental methods. We sequenced our library at the Bauer Core Facility of the FAS Center for Systems Biology at Harvard University (Cambridge, MA), using one lane of an Illumina HiSeq 2500 Rapid Run flow cell with 150 base pair paired‐end sequencing.
10
Shotgun Metagenomics Library Preparation
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Genomic DNA was extracted via MP FastDNA SPIN kits (Solon, Ohio) with a minor modification that included three freeze-thaw cycles (McNamara et al., 2014) . The concentrations of the extracted DNA were measured using NanoDrop Lite Spectrophotometer (Thermo-Fisher Scientific, MA), a compact version of NanoDrop 2000/2000C (Desjardins and Conklin, 2010) , and were sent to a sequencing facility at the Research Technology Supporting Facility (RTSF) in Michigan State University after adjusting the concentrations to 10 ng/μl by diluting with DNase free water. Shotgun metagenomics library was prepared using TruSeq Nano DNA Library Preparation Kit (illumine, CA) with insertion size of approximately 550bp long at the RTSF. Adapter sequences were ligated and purified according to the TruSeq Nano DNA Library Preparation Kit protocol (Illumina, 2015) . The concentration of each library was measured using Qubit and Kapa Library Quantification qPCR and libraries were pooled in equimolar amounts. Sequencing was performed using the Illumina HiSeq 2500 Rapid Run Flow Cell in 2 x 150 paired end format with dual lane loading. The sequencing of the same library pool was repeated with a single lane to generate more sequence reads. The raw read data were demultiplexed and converted to fastq format.
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