Two independent primary AML lines per oncogene (CALM/AF10 or MLL/AF9) were obtained from the bone marrow of leukemic mice (CALM/AF10 #1 and #2 and MLL/AF9 #1 and #2; Figure S2A ) and RNA was extracted using an RNeasy Mini Kit (Qiagen). Libraries were generated using the Ovation Mouse RNA-Seq System following the manufacturer’s specifications (NuGEN). All libraries underwent 75 bp single-end sequencing on an Illumina NextSeq500 sequencer. For RNA-seq of RG3039-treated AML cells (Figure 4 ), CALM/AF10 cells or GMPs (Lin−Sca-1−c-Kit+FcγR+CD34+) were treated with RG3039 (1 μM) in the presence of cytokines, harvested over a 0, 6 and 10h time course, and RNA samples were prepared. Libraries were constructed and underwent 150 bp paired-end sequencing on an Illumina NextSeq500 sequencer.
Nextseq 500 sequencer
Manufactured by Illumina
Sourced in United States, Germany, France, Italy, Canada, United Kingdom
The NextSeq 500 is a high-throughput DNA sequencing instrument manufactured by Illumina. It is designed to perform next-generation sequencing of DNA samples. The NextSeq 500 utilizes Illumina's sequencing-by-synthesis technology to generate sequencing data.
Automatically generated - may contain errors
Lab products found in correlation
Rneasy mini kit, by Qiagen (43 mentions)
2100 bioanalyzer, by Agilent Technologies (28 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (26 mentions)
Qubit dsdna hs assay kit, by Thermo Fisher Scientific (26 mentions)
Trizol reagent, by Thermo Fisher Scientific (25 mentions)
Bioanalyzer, by Agilent Technologies (20 mentions)
Trizol, by Thermo Fisher Scientific (18 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (18 mentions)
Ampure xp bead, by Beckman Coulter (17 mentions)
Nextera xt dna sample preparation kit, by Illumina (16 mentions)
Smart seq v4 ultra low input rna kit, by Takara Bio (15 mentions)
Nextseq 500 550 high output kit v2, by Illumina (15 mentions)
Truseq stranded mrna sample preparation kit, by Illumina (15 mentions)
Tapestation, by Agilent Technologies (14 mentions)
Agencourt ampure xp bead, by Beckman Coulter (14 mentions)
Nextera xt library prep kit, by Illumina (13 mentions)
Kapa library quantification kit for platform, by Illumina (12 mentions)
Kapa library quantification kit, by Roche (12 mentions)
Nanodrop, by Thermo Fisher Scientific (12 mentions)
Ampure xp kit, by Beckman Coulter (12 mentions)
849 protocols using nextseq 500 sequencer
1
Transcriptome Analysis of Leukemic Cell Lines
2
Cloning and Sequencing of p19-Captured Small dsRNAs
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p19-captured small dsRNAs isolated from E. coli cells expressing p19 from a plasmid were cloned and sequenced according to Huang et al. (33 (link)). p19-captured small dsRNAs (for E and S samples), isolated from E. coli cells with integrated His-tagged p19, were cloned using the NEBNext small RNA library prep set for SOLiD (E6160; NEB) according to the manufacturer’s protocol and sequenced on a SOLiD sequencer at NEB. E-R1, E-R2, S-R1, and S-R2 samples were cloned using the NEBNext multiplex small RNA library prep set for Illumina (E7300S; NEB) according to the manufacturer’s protocol and sequenced on an Illumina NextSeq500 sequencer. Small RNAs from RNase III and Dicer digestion assays were cloned using the NEBNext small RNA library prep set for Illumina (E7330L; NEB) according to the manufacturer’s protocol and sequenced on an Illumina GAII sequencer at NEB or on an Illumina NextSeq 500 sequencer.
3
Metagenome and Metatranscriptome Sequencing of Environmental Samples
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Metagenome sequencing libraries were prepared with the NEBNext Ultra DNA Library Prep Kit for Illumina (New England Biolabs GmbH, Frankfurt am Main, Germany) and sequenced on a NextSeq500 sequencer (Illumina) using 2 × 150 bases. In addition, DNA from November 2017 was sequenced by PacBio sequencing on a Sequel instrument (Pacific Biosciences, Menlo Park, CA, USA) using circular consensus sequencing with a target length of 2 kilobases. Bioinformatics processing, genome binning, and phylogenetic analysis followed standard procedures as described in the Supplementary Text.
For metatranscriptome sequencing, messenger RNA (mRNA) was enriched from total RNA extracts by depleting ribosomal RNA with the Ribo-off rRNA Depletion Kit for bacteria (Vazyme, Nanjing, China). Thereafter, the sequencing library was prepared with the TruSeq Stranded mRNA Library Prep (Illumina) and sequenced on a NextSeq500 sequencer using 2 × 150 bases. Bioinformatics processing to determine the transcriptional levels of selected genes as well as network analysis of co-transcribed genes are detailed in the Supplementary Text.
For metatranscriptome sequencing, messenger RNA (mRNA) was enriched from total RNA extracts by depleting ribosomal RNA with the Ribo-off rRNA Depletion Kit for bacteria (Vazyme, Nanjing, China). Thereafter, the sequencing library was prepared with the TruSeq Stranded mRNA Library Prep (Illumina) and sequenced on a NextSeq500 sequencer using 2 × 150 bases. Bioinformatics processing to determine the transcriptional levels of selected genes as well as network analysis of co-transcribed genes are detailed in the Supplementary Text.
4
Single-cell RNA-seq on Tumor Samples
5
Prenatal Diagnostics Using rES
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Upon inclusion, patients followed routine prenatal diagnostics starting with QF‐PCR for detection of trisomies 13, 18, and 21, monosomy X and triploidy. Following normal QF‐PCR results, routine‐care SNP‐array and rES were started in parallel (Figure 1 for study workflow).
Fetal DNA was extracted from chorionic villi or amniotic fluid and parental DNA from peripheral blood using standard diagnostic procedures. Identification of the sample and exclusion of maternal cell contamination in the fetal DNA was carried out using QF‐PCR data of the fetus and the mother. Fetal and parental DNA were prepared for rES using SureSelect Human All Exon V6 (Agilent) target enrichment, according to standard procedures, on Bravo automated liquid handling robots (Agilent), and then sequenced on an Illumina NextSeq500 sequencer aiming for 20x coverage for 95% of the target genes. Fetal DNA was prepared for rES in duplo and sequenced in two separate runs for validation purposes in order to avoid time‐consuming Sanger sequencing validation. Two automated DNA isolation systems (Maxwell, Promega), two automated library and enrichment robots (Bravo, Agilent), two TapeStation systems (Agilent) for measuring DNA concentrations, two NextSeq500 sequencers (Illumina) and two data analysis clusters ensured redundancy in case of failure or malfunctions.
Fetal DNA was extracted from chorionic villi or amniotic fluid and parental DNA from peripheral blood using standard diagnostic procedures. Identification of the sample and exclusion of maternal cell contamination in the fetal DNA was carried out using QF‐PCR data of the fetus and the mother. Fetal and parental DNA were prepared for rES using SureSelect Human All Exon V6 (Agilent) target enrichment, according to standard procedures, on Bravo automated liquid handling robots (Agilent), and then sequenced on an Illumina NextSeq500 sequencer aiming for 20x coverage for 95% of the target genes. Fetal DNA was prepared for rES in duplo and sequenced in two separate runs for validation purposes in order to avoid time‐consuming Sanger sequencing validation. Two automated DNA isolation systems (Maxwell, Promega), two automated library and enrichment robots (Bravo, Agilent), two TapeStation systems (Agilent) for measuring DNA concentrations, two NextSeq500 sequencers (Illumina) and two data analysis clusters ensured redundancy in case of failure or malfunctions.
6
Transcriptome Sequencing from RNA Samples
7
Targeted Exome Sequencing for Genetic Phenotypes
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Following genomic DNA extraction, qualified DNA sample was randomly sheared to generate 180–280 bp DNA fragments, which were selected for the preparation of DNA libraries. The currently identified 6259 genetic phenotypes by OMIM were detected, and a total of 1839 genes associated with clinical phenotypes of the proband were focused. The libraries were hybridized with biotin-labeled probes in liquid phase; then the streptavidin magnetic beads were used to bind with biotin-containing target fragments for the capture of the exons of these genes. The paired-end reads of 150 bp sequencing was performed on an Illumina NextSeq 500 sequencer (Illumina, San Diego, CA, USA) after enrichment and quality inspection of the libraries.
8
Cultivation and Characterization of Neisseria meningitidis Strains
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Bacteria were grown overnight on Columbia agar plates with 5% sheep blood (bioMérieux) at 37°C in 5% CO2. On the next day, a fraction of the bacteria was transferred to 10 ml proteose peptone medium (PPM) supplemented with 1× Kellogg´s supplement, 0.01 M MgCl2, and 0.005 M NaHCO3 (PPM+) and incubated for 90 min at 37°C in an orbital shaker at 200 rpm. Neisseria meningitidis strain MC58 is a serogroup (Sg) B (MenB) isolate (United Kingdom, 1983) of the clonal complex (cc) 32 and was kindly provided by E. R. Moxon (Tettelin et al., 2001 (link)). N. meningitidis Sg C (MenC) strain 8013/clone12 (cc18) was kindly provided by M. Taha (Nassif et al., 1993 (link)). N. meningitidis SgB (MenB) strain α711 (cc32) and N. meningitidis SgW (MenW) strain α275 were isolated during the Bavarian meningococcal carriage study (Claus et al., 2005 (link); Harrison et al., 2013 (link)). N. meningitidis SgW (MenW) strain DE13664 was isolated from CSF. Meningococcal strains DE13664 and α711 were subject to whole-genome sequencing. Genome sequencing was performed on an Illumina NextSeq 500 sequencer (Illumina Inc., San Diego, CA, USA) at the core unit Systems Medicine of the University of Würzburg. The resulting FASTQ files were de novo assembled using the Velvet assembler integrated in Ridom SeqSphere+ software (Ridom GmbH, Münster, Germany). All strains are summarized in Table 1
9
Metagenomic Analysis of Stool Samples
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Stool samples were collected at different time points per individual (Supplementary Table 3 ). DNA was extracted from ~50 mg of stool samples in two stages: an initial homogenization in Lysis Matrix E tubes (MP Biomedicals) with a Precellys 24 Tissue Homogenizer (Bertin Instruments) and processing of the resultant supernatant using the MagAttract PowerMicrobiome DNA/RNA EP kit (Qiagen) on an Eppendorf automated liquid handling system as per the manufacturer’s instructions.
Isolated DNA was checked for concentration and quality on a BioTek Synergy HTX plate reader.
Metagenomic libraries were prepared using the Nextera DNA Flex Library Prep Kit (Illumina) per the manufacturer’s instructions with 100 ng of DNA as sample input. The concentration of the libraries was quantified using the Qubit dsDNA HS assay on a Qubit 2.0 fluorometer (Life Technologies). Library size and quality were assessed via the Agilent High Sensitivity D5000 ScreenTape on an Agilent 4200 Tapestation.
Metagenomic libraries were normalized to an equimolar concentration and pooled. The pool was diluted to 1.8 pM, mixed with a 1% PhiX control library and paired-end sequenced (2 × 75 bp) using a NextSeq 500/550 High Output v2 150-cycle Reagent Cartridge on a NextSeq 500 sequencer (Illumina).
Isolated DNA was checked for concentration and quality on a BioTek Synergy HTX plate reader.
Metagenomic libraries were prepared using the Nextera DNA Flex Library Prep Kit (Illumina) per the manufacturer’s instructions with 100 ng of DNA as sample input. The concentration of the libraries was quantified using the Qubit dsDNA HS assay on a Qubit 2.0 fluorometer (Life Technologies). Library size and quality were assessed via the Agilent High Sensitivity D5000 ScreenTape on an Agilent 4200 Tapestation.
Metagenomic libraries were normalized to an equimolar concentration and pooled. The pool was diluted to 1.8 pM, mixed with a 1% PhiX control library and paired-end sequenced (2 × 75 bp) using a NextSeq 500/550 High Output v2 150-cycle Reagent Cartridge on a NextSeq 500 sequencer (Illumina).
10
H3K4me3 and H3K27me3 ChIP-seq
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ChIP-seq was performed as described previously19 (link),46 (link). Briefly, crosslinked chromatin was fragmented by Micrococcal nuclease digestion and immunoprecipitated with anti-H3K4me3 or anti-H3K27me3 antibodies (Abcam, Cambridge, MA). After ChIP, the crosslinking was reversed and DNA fragments were purified. The sequencing library was constructed using Illumina’s TruSeq sample preparation kit according to the provided instruction. The samples before immunoprecipitation served as input controls. Sequencing was performed using Illumina Nextseq 500 sequencer.
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