We collected fresh and healthy leaves of four species (L. spinosa, S. bogneri, Z. zamiifolia, and O. aquaticum) from the Araceae Greenhouse at the Missouri Botanical Garden in St. Louis, Missouri (Figure 1 ). Whole genomic DNA was extracted from the collected leaves using Qiagen DNeasy Minikit (Qiagen, Germantown, MD, USA), with some modifications following a previous approach [11 (link),19 (link)]. DNA quality and quantity were confirmed using 1% agarose gel electrophoresis and Nanodrop (Thermo Fisher Scientific, Waltham, MA, USA). The libraries were constructed following the manufacturer’s protocol for Illumina TruSeq kits (Illumina, Inc., San Diego, CA, USA) in the Pires laboratory at the University of Missouri, Columbia. The Illumina HiSeq 2000 platform was used to sequence qualified libraries from single end with 100 bp short reads at the University of Missouri DNA Core.
Truseq kits
Manufactured by Illumina
Sourced in United States
The TruSeq kits are a suite of library preparation products developed by Illumina. They provide a streamlined workflow for preparing DNA or RNA samples for sequencing on Illumina's next-generation sequencing platforms. The kits contain reagents and protocols for sample fragmentation, adapter ligation, and amplification, enabling users to generate sequencing-ready libraries from a variety of input materials.
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Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (9 mentions)
Nanodrop, by Thermo Fisher Scientific (8 mentions)
Trizol, by Thermo Fisher Scientific (6 mentions)
Hiseq 2000 platform, by Illumina (5 mentions)
Agilent bioanalyzer, by Agilent Technologies (5 mentions)
Dneasy mini kit, by Qiagen (4 mentions)
Hiseq 2000 sequencer, by Illumina (4 mentions)
Hiseq 2000, by Illumina (4 mentions)
Rneasy mini kit, by Qiagen (4 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (3 mentions)
Direct zol rna miniprep, by Zymo Research (3 mentions)
Ribo zero, by Illumina (3 mentions)
Direct zol, by Zymo Research (2 mentions)
Casava v1.8.2 software tools, by Illumina (2 mentions)
Ribo zero column, by Illumina (2 mentions)
Scriptseq v2, by Illumina (2 mentions)
Superscript 3, by Thermo Fisher Scientific (2 mentions)
Novaseq 6000, by Illumina (2 mentions)
Truseq rna method, by Illumina (2 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (2 mentions)
44 protocols using truseq kits
1
Genomic DNA Extraction and Sequencing
2
Transcriptome Analysis of Aging Hippocampus
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Transcriptomic (RNA-seq) and gene expression analyses were performed on isolated whole hippocampus (an area of the brain associated with cognitive function/memory that deteriorates with brain aging and tauopathy) via methods previously described (Wahl et al., 2022) (link). Frozen samples (n = 4 per group; 2M/2F) were transferred into Trizol (ThermoFisher) and homogenized on ice (SP Bel-Art ProCulture Micro-Tube Homogenizer System; VWR Scientific). RNA was isolated using an RNA-specific spin column kit (Direct-Zol, Zymo Research) and treated with DNase to remove genomic DNA. Poly(A)-selected libraries were generated using magnetic oligo dT beads (ThermoFisher) and Illumina TruSeq kits, and libraries were sequenced on an Illumina NovaSeq 6000 platform to generate an average of 66,000 150-bp paired-end reads per sample. Reads were trimmed and filtered with the fastp program (Chen et al., 2018) (link), then aligned to the mm10 Mus musculus genome using the STAR aligner (Dobin et al., 2013) (link), and the mapping rates were ≥70 % uniquely mapped reads per sample. Differential gene expression was analyzed with DESeq2 and genes/transcripts with BaseMean read counts of <10 were filtered and removed from downstream analyses (Bourgon et
3
Whole Genome Sequencing of Araceae
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We collected fresh and healthy leaves of four species (L. spinosa, S. bogneri, Z. zamiifolia, and O. aquaticum) from the Araceae Greenhouse at the Missouri Botanical Garden in St.
Louis, Missouri. Whole genomic DNA was extracted from the collected leaves using Qiagen DNeasy Minikit (Qiagen, Germantown, Maryland, USA) with some modification following a previous approach [10, 18] . DNA quality and quantity were confirmed by 1% agarose gel electrophoresis and Nanodrop (ThermoScientific, Delaware, USA). The libraries were constructed following manufacturer's protocol of Illumina TruSeq kits (Illumina, Inc., San Diego, California) in the Pires laboratory at the University of Missouri, Columbia. The Illumina HiSeq 2000 platform was used to sequence qualified libraries from single end with 100 bp short reads at the University of Missouri DNA Core.
Louis, Missouri. Whole genomic DNA was extracted from the collected leaves using Qiagen DNeasy Minikit (Qiagen, Germantown, Maryland, USA) with some modification following a previous approach [10, 18] . DNA quality and quantity were confirmed by 1% agarose gel electrophoresis and Nanodrop (ThermoScientific, Delaware, USA). The libraries were constructed following manufacturer's protocol of Illumina TruSeq kits (Illumina, Inc., San Diego, California) in the Pires laboratory at the University of Missouri, Columbia. The Illumina HiSeq 2000 platform was used to sequence qualified libraries from single end with 100 bp short reads at the University of Missouri DNA Core.
4
Hybrid Genome Assembly for Gram-positive Bacteria
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DNA was isolated with the MasterPure Gram-positive DNA purification kit (Epicenter Biosciences). Illumina sequencing libraries were prepared by using either Nextera or TruSeq kits with indexed-encoded adapters from Illumina, according to the manufacturer’s instructions. Libraries were pooled for sequencing on Illumina GAIIx or HiSeq, and paired-end sequence reads were obtained representing 50- to 200-fold genome coverage. Strains representing two distinct ST2 lineages (UH9907 and UH10707) were also subjected to SMRT sequencing on a PacBio. PacBio sequencing resulted in long reads (median, ~3.5 kbp) and ~10× to 20× coverage of error-corrected reads. Illumina sequence data were assembled by using Velvet (63 ). A range of k-mer values was evaluated, and the assembly with the largest N50 was selected for annotation and analysis. The PacBio sequence was assembled by using the HGAP (64 (link)). Several hybrid assemblies combining PacBio with Illumina sequence data were not sufficiently superior to the assembly obtained by the HGAP (data not shown). For information on assembly quality, see Table S3 in the supplemental material. Assemblies of Illumina data generally had contig N50 values of >100 kbp. The PacBio assemblies had contig N50 values of >1 Mbp.
5
Illumina RNA-seq Library Preparation
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Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. RNA concentration and quality were assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Equal amount of RNA samples from three biological replicates within each line each treatment group were pooled in preparation to construct standard cDNA libraries using Illumina TruSeq kits and reagents following the manufacturer’s protocol for deep sequencing. The libraries were sequenced on an Illumina HiSeq2000 sequencer for single end 50 base sequencing run. The post sequencing processes, including image analysis, base calling, and Q-Score calculation, were carried out using Real Time Analysis (v1.13.48); read demultiplexing and conversion to final FASTQ files, using CASAVA (v1.8.2) software tools (Illumina Inc., San Diego, CA, USA). The library preparation, RNA sequence read extraction, and preliminary read quality control were performed at the Research Technology Support Facility, Michigan State University.
6
Poly(A) and Total RNA Sequencing
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RNA for poly(A) and total RNA sequencing libraries was extracted from whole animals by TRIzol extraction. Genomic DNA was removed using TURBO DNase (Invitrogen). For total RNA libraries, 5 μg of RNA was run through a Ribo-Zero column (Epicenter, #R2C1046) to remove ribosomal RNA. For poly(A)-selected libraries, RNA was selected using sera-mag magnetic oligo dT beads (Therma Scientific). Libraries were created using Illumina TruSeq kits (RS-122-2001). RNA recovered by immunoprecipitation with the J2 antibody (three biologically independent lysates) of young adult worms as well as input material (as a loading control) was converted into strand-specific total RNA libraries using V2 Scriptseq (Epicenter #SSV21106) kits following manufacturer's instructions, except reverse transcription was done with SuperScript III (Invitrogen #18080-044) using incrementally increasing temperatures from 42 to 59°C to allow for transcription though structured RNAs. rRNA was not removed from J2-IP RNA samples. Immunoprecipitated DNA from ChIP samples was converted in sequencing libraries using ChIP-Seq DNA Sample Prep Kit (IP-102–1001). Libraries were sequenced on Illumina HiSeq 2000 platforms. Data were deposited under GEO accession number GSE61581.
7
Genomic DNA Extraction and Sequencing of Aroids
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We collected fresh healthy leaves of P. scandens and A. huixtlense from the Aroid Greenhouse at the Missouri Botanical Garden in St. Louis, Missouri. Total genomic DNA was extracted from these leaves using a Qiagen DNeasy Minikit (Qiagen, Germantown, Maryland, USA) following Henriquez et al. (2020a (link)). Confirmation of the quality and quantity of DNA was performed using 1% gel electrophoresis and Nanodrop (ThermoScientific, Delaware, USA). Library preparation and sequencing were performed using TruSeq kits (Illumina, Inc., San Diego, California) in the Pires lab at the University of Missouri, Columbia following Henriquez et al. (2020a (link)).
8
Transcriptional Profiling of p73 in MTECs
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Gene expression for wild-type and p73 knockout MTECs at ALI D0, D4, D7, and D14 was measured. RNA was isolated with RNeasy kit (Qiagen). RNA-seq and sRNA-seq libraries were prepared by TruSeq kits (Illumina). Library quality and sample concentration were checked before sequencing (50-base-pair single end) on a HiSeq 2000 (Illumina) using TruSeq SR cluster kit version 3-cBot-HS and TruSeq SBS kit version 3-HS. All experiments ran in triplicate, sequencing three biological replicates per condition. Murine RNA-seq data were aligned to the Mus musculus mm10 genome using STAR aligner (version 2.3.0e_r291) with default options. Read counts for all genes and all exons (Ensembl annotation version 72) were obtained using FeaturesCount (version 1.4.6) (Liao et al. 2014 (link)). To identify enriched GO categories, the Web service WebGestalt was used. GO category enrichment was assessed by calculating the fold change between observed and expected numbers of genes of a given GO category, where terms were scored enriched if they had an adjusted P-value of <0.1 (GEO no. GSE75717).
9
Comprehensive Genome Profiling of Diagnostic Samples
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The mapping results were compared to the known aberrations from karyotyping, FISH (Fluorescence in situ hybridization) analysis, array-CGH and RNA-panel sequencing (methods are described in [23 (link)]). Whole transcriptome sequencing (RNAseq) of diagnostic samples (in 5/12 patients, #1–5) was performed as a service at the Sequencing Core Facility of the Max Planck Institute for Molecular Genetics (Berlin, Germany) (Table 1 ). Sequencing libraries were prepared from DNA and total RNA using TruSeq kits (Illumina, San Diego, CA, USA) and sequenced on HiSeq2000 platform (Illumina, San Diego, CA, USA). The read pairs were aligned to the human genome reference hg19 using STAR (RNAseq) aligners and further processed by Picard tools (http://broadinstitute.github.io/picard/releases/tag/1.113 accessed on 29 August 2021).
We focus here on called SVs and CNVs, which are known to be clinically relevant or which may be clinically relevant based on their frequency in reference genomes but we will not address polymorphic regions according to DGV (http://dgv.tcag.ca/dgv/app/home , release date 25 February 2021). A selected number of SVs called in Bionano, but undetected by standard methods were validated by breakpoint-spanning PCR or FISH.
We focus here on called SVs and CNVs, which are known to be clinically relevant or which may be clinically relevant based on their frequency in reference genomes but we will not address polymorphic regions according to DGV (
10
RNA-seq Analysis of Mouse Brain Aging
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