For the sifakas housed at the DLC, we extracted DNA using the QIAGEN Blood and Cell Culture Kit. For the wild Verreaux’s sifaka individual, we extracted DNA as described in (107 (link)) from banked ear tissue. All samples then underwent quality control via PicoGreen quantification and gel imaging for concentration and purity. We used the manufacturer’s recommended procedures to generate Illumina 100-bp paired-end whole-genome sequencing libraries. We sequenced those libraries to 104.7× whole-genome coverage via runs of 12 lanes on an Illumina Hi-Seq 2000 instrument and 1 lane on an Illumina Hi-Seq 2500 instrument for the individual used for the assembly. The additional individuals were sequenced on two lanes of an Illumina Hi-Seq 2000 instrument each. For the Verreaux’s sifaka from Bezà Mahafaly Special Reserve, 150-bp paired-end libraries were generated and sequenced on a single Illumina Hi-Seq X lane.
Hiseq 2000 instrument
Manufactured by Illumina
Sourced in United States, China, Poland
The HiSeq 2000 is a high-throughput DNA sequencing instrument manufactured by Illumina. It is capable of generating large volumes of DNA sequence data through parallel processing of multiple samples. The core function of the HiSeq 2000 is to perform next-generation DNA sequencing using Illumina's proprietary sequencing-by-synthesis technology.
Automatically generated - may contain errors
Lab products found in correlation
Agilent 2100 bioanalyzer, by Agilent Technologies (21 mentions)
Trizol reagent, by Thermo Fisher Scientific (12 mentions)
2100 bioanalyzer, by Agilent Technologies (7 mentions)
Truseq rna sample preparation kit v2, by Illumina (6 mentions)
Truseq rna sample preparation kit, by Illumina (6 mentions)
Hiseq 2000, by Illumina (6 mentions)
Truseq small rna sample prep kit, by Illumina (6 mentions)
Bioanalyzer, by Agilent Technologies (5 mentions)
Cfx96 instrument, by Bio-Rad (5 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (4 mentions)
Nebnext ultra directional rna library prep kit, by Illumina (4 mentions)
Ribo zero magnetic gold kit, by Illumina (4 mentions)
Truseq nano kit, by Illumina (3 mentions)
Sureselect custom kit, by Agilent Technologies (3 mentions)
Rneasy plus mini kit, by Qiagen (3 mentions)
Rneasy plant mini kit, by Qiagen (3 mentions)
Ribo zero rrna removal kit h m r, by Illumina (3 mentions)
Dneasy plant mini kit, by Qiagen (3 mentions)
Kapa library quant kit, by Roche (3 mentions)
Hiseq 2500 instrument, by Illumina (2 mentions)
145 protocols using hiseq 2000 instrument
1
Whole-Genome Sequencing of Sifaka Primates
2
Streptochaeta miRNA Annotation and Validation
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To annotate microRNAs (miRNAs) present in the Streptochaeta genome, we (i) sequenced sRNAs from leaf, anther and pistil tissues, (ii) compared miRNAs present in anthers to those of three other representative monocots (rice, maize, and asparagus), and (iii) validated gene targets of these miRNAs.
We collected tissues from leaf and pistil as well as 1.5, 3, and 4 mm anthers. Samples were immediately frozen in liquid nitrogen and kept at –80°C prior to RNA isolation. Total RNA was isolated using the PureLink Plant RNA Reagent (Thermo Fisher Scientific, Waltham, MA, United States). sRNA libraries were published previously (Patel et al., 2021 (link)). RNA sequencing libraries were prepared from the same material using the Illumina TruSeq stranded RNA-seq preparation kit (Illumina Inc., United States) following manufacturer’s instructions. Parallel analysis of RNA ends (PARE) libraries were prepared from a total of 20 μg of total RNA following the method described by Zhai et al. (2014) (link). For all types of libraries, single-end sequencing was performed on an Illumina HiSeq 2000 instrument (Illumina Inc., United States) at the University of Delaware DNA Sequencing and Genotyping Center.
We collected tissues from leaf and pistil as well as 1.5, 3, and 4 mm anthers. Samples were immediately frozen in liquid nitrogen and kept at –80°C prior to RNA isolation. Total RNA was isolated using the PureLink Plant RNA Reagent (Thermo Fisher Scientific, Waltham, MA, United States). sRNA libraries were published previously (Patel et al., 2021 (link)). RNA sequencing libraries were prepared from the same material using the Illumina TruSeq stranded RNA-seq preparation kit (Illumina Inc., United States) following manufacturer’s instructions. Parallel analysis of RNA ends (PARE) libraries were prepared from a total of 20 μg of total RNA following the method described by Zhai et al. (2014) (link). For all types of libraries, single-end sequencing was performed on an Illumina HiSeq 2000 instrument (Illumina Inc., United States) at the University of Delaware DNA Sequencing and Genotyping Center.
3
Genotyping of Soybean RILs
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Genomic DNA was extracted from healthy young leaves of IT208075 and the 235 F7:8 RILs using the GenoAll® ExgeneTM Plant SV kit (GeneAll Biotechnology, Seoul, Korea). DNA quality was assessed by the 260/280 nm ratio using a Nanodrop 3000 spectrometer (Thermo Scientific, Wilmington, DE, USA). DNA was quantified using the Invitrogen Quant-iT PicoGreen® dsDNA Assay kit (Life Technologies, Burlington, ON, Canada), and its concentration was adjusted to 20 ng/μL. IT208075 was re-sequenced with 9× sequencing depth on an Illumina HiSeq2000 platform (Illumina Inc., San Diego, CA, USA). The Illumina reads were deposited in the database of the National Center for Biotechnology Information (NCBI) under BioProject accession number PRJNA698712 . The genotyping-by-sequencing (GBS) method was used to genotype the RILs (Elshire et al., 2011 (link)). After digestion with the restriction enzyme ApeKI, a GBS library was constructed following the procedure described previously (Hwang et al., 2017 (link); Yoon et al., 2019 (link)); the barcode adapters used in this study are listed in Supplementary Table 1 . Three separate libraries were constructed using pooling amplified DNA samples from 84 or 85 RILs for each library. Single-end sequencing of the GBS libraries was performed on 3 lanes of an Illumina HiSeq2000 instrument (Illumina Inc.).
4
Transcriptome Analysis of Lemna gibba
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Fronds grown on inorganic nitrogen or on organic nitrogen medium were harvested and immediately frozen in liquid nitrogen. For each condition three biological replicates were analyzed. Total RNA was extracted by grinding 200 mg frozen tissue to a fine powder in liquid nitrogen with a mortar and pestle, followed by extraction with TRIzol Reagent (Invitrogen, MA, USA) and ethanol precipitation. Total RNA was resuspended and treated with RQ1 RNase-free DNase (Promega,WI, USA) on an RNA Clean & Concentrator-5 column (Zymo, CA, USA). The purity and concentration of RNA were determined by a NanoDrop Nd-1000 spectrophotometer (Thermo Scientific, MA, USA) and a Qubit fluorometer (Qiagen, MD, USA). Polyadenylated RNA was enriched from total RNA with the Dynabeads mRNA Purification Kit (Life Technologies, CA, USA). Indexed, directional RNA-seq libraries were prepared with ScriptSeq v2 reagents (Epicentre, WI, USA) from 40–50 ng of polyA-selected RNA according to the manufacturer’s instructions. Libraries were sequenced on an Illumina HiSeq 2000 instrument (Illumina, Inc., CA, USA) generating 101 bp paired-end reads. Reads from each sample were mapped against the 21,830 predicted transcripts of the Lemna gibba draft genome v0.5.1 [80 ] using Bowtie [81 (link)].
5
Transcriptomic Analysis of Sorted Subpopulations
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For quality control the integrity of the RNA was assayed at several steps during the staining and sorting procedure with the aid of the Aurum Total RNA kit (Bio-Rad, Copenhagen, Denmark) and the Agilent 2100 Bioanalyzer (Agilent Technologies, Glostrup, Denmark) both as prescribed by the manufacture (Fig. 1a ).
After flash-freezing the transcriptomic analysis of the frozen subpopulations (SP1–4) was performed by AROS Applied Biotechnology (Arhus, Denmark) using the SMART-Seq v4 Ultra Low Input RNA Kit for cDNA and Low Input Library Prep Kit V2 for sequencing library preparation (both by Takara Bio, Otsu, Japan), and Illumina HiSeq 2000 instrument (Illumina Inc., San Diego, CA) for sequencing. The sequencing data were imported as paired-ends into CLC Genomic Workbench v9.0.1 (CLC bio, Aarhus, Denmark) to enable RNA-seq reports, including reads average PHRED score and mapping statistics against the annotated Homo sapiens UCSC hg19 reference genome (http://hgdownload.cse.ucsc.edu/goldenPath/hg19/bigZips/ ). The alignment quality was further assessed by the Qualimap v2.2 (http://qualimap.bioinfo . cipf.es/) producing the read coverage and GC content profiles [23 (link)].
After flash-freezing the transcriptomic analysis of the frozen subpopulations (SP1–4) was performed by AROS Applied Biotechnology (Arhus, Denmark) using the SMART-Seq v4 Ultra Low Input RNA Kit for cDNA and Low Input Library Prep Kit V2 for sequencing library preparation (both by Takara Bio, Otsu, Japan), and Illumina HiSeq 2000 instrument (Illumina Inc., San Diego, CA) for sequencing. The sequencing data were imported as paired-ends into CLC Genomic Workbench v9.0.1 (CLC bio, Aarhus, Denmark) to enable RNA-seq reports, including reads average PHRED score and mapping statistics against the annotated Homo sapiens UCSC hg19 reference genome (
6
Nextera Transposon-Based DNA Sequencing
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From each viral pool, 75 ng of viral DNA was sheared and ligated to adaptors by transposition using the Nextera kit (Epicentre), following the manufacturer's direction with several modifications: transposition was performed in 20 ul volume using 0.1 µl Nextera transposase enzyme and “HMW” reaction buffer at final 1X. Transposition was carried out for 10 minutes at 55°C, after which sheared, adaptor-ligated templates were transferred directly to PCR reactions for amplification. In addition, PCR incorporated a sample-specific barcode tag on the reverse primer. Amplified libraries were pooled and cleaned using Ampure beads (Beckman Coulter) at a 1.8∶1 ratio of beads to input. The pooled libraries were sequenced on an Illumina HiSeq 2000 instrument (Illumina) using 101-bp forward and reverse reads with a 9-bp index to read the per-sample barcode.
7
RNA-seq of FFPE Specimens Protocol
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RNA-seq of FFPE specimens was done as previously reported [14 (link)]. Briefly, FFPE blocks were sectioned (10μM thickness), RNA isolated using the Allprep RNA/DNA FFPE kit (Qiagen), and RNA quality verified by Agilent Bioanlyzer. Sequencing libraries (150 bp average insert size) were then prepared from 100 ng of rRNA-depleted RNA using TruSeq RNA Sample Preparation Kit v2 (Illumina), with four indexed libraries loaded per flow-cell lane. Paired-end 75-bp sequencing was carried out on a HiSeq 2000 instrument (Illumina), with the three AcCC libraries yielding 103–110 million sequencing reads. For discovery of gene fusions, evidenced by paired reads mapping to two different genes, and/or reads spanning predicted fusion junctions, we used Chimeriscan [15 (link)], TopHat-Fusion [16 (link)], SnowShoes-FTD [17 (link)], and deFuse [18 (link)] software. RNA-seq reads are available at GEO (accession GSE76354). Analysis of publically-available The Cancer Genome Atlas (TCGA) RNA-seq data was done using Wanderer [19 (link)], a web-based interactive viewer.
8
Chromatin Immunoprecipitation Sequencing Protocol
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ChIP was performed as described before (Galbraith et al. 2013 (link)) and described in detail in the Supplemental Methods . Briefly, after drug or vehicle treatment, cells were cross-linked using 1% formaldehyde, washed twice with cold PBS, lysed in RIPA buffer, and cell lysates sonicated. Following immunoprecipitation with anti-TP53 antibody, immune complexes were washed, bound proteins eluted, and cross-linking reversed prior to DNA purification. Sequencing libraries were prepared using Illumina's TruSeq ChIP Library Preparation kit, and barcoded libraries sequenced on an Illumina HiSeq2000 instrument.
9
Exome Sequencing and Variant Filtering
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Genomic DNA was isolated from venous blood or saliva obtained from the cases at enrolment or from archived samples. DNA library capture was performed using ROCHE NimbleGen SeqCap EZ 64 Mb Human Exome Library version 3.0 (ROCHE NimbleGen, Inc., Madison, WI, USA). The libraries were sequenced on an Illumina Hiseq 2000 instrument (Additional file 5 [25 (link)-27 (link)]). In order to filter for technical artefacts and variants unlikely to be pathogenic, variants were excluded from further analysis if they fulfilled any of the following criteria: (1) variant allele frequency >0.1% in any of the reference cohorts (Additional file 6 ); (2) variant not predicted to alter protein by snpEff 3.4 [28 (link)]; (3) variant not present in other affected pedigree members recruited to BRIDGE-BPD; (4) <3 reads supporting the alternate allele; (5) allele count >1 among 20 in-house whole-genome sequenced controls; (6) overall allele count >20 including exomes from other BRIDGE projects.
10
Total RNA Extraction and Sequencing
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