Pretreatment blood or bone marrow samples were studied centrally by G-and R-banding analysis. Chromosomal abnormalities were described according to the International System for Human Cytogenetic Nomenclature.18 DNA was extracted as described before using the Allprep DNA/RNA purification kit (Qiagen, Hilden, Germany).19 (link) DNA sequencing libraries were prepared from samples at diagnosis (n=116) and at relapse (n=20) with a custom TruSight myeloid sequencing panel according to the manufacturers’ instructions (Illumina, San Diego, CA), which included 46 entire genes or hotspots recurrently found in myeloid leukemias (Supplementary Table S1). All samples received individual dual indexes and were pooled at equimolar concentrations. Eighty samples per lane were sequenced on an Illumina HiSeq2500 sequencer using the HiSeq Rapid SBS Kit v2 (Illumina, San Diego, CA) for 251 cycles in both directions. The sequencing data was analysed as described before with modifications as detailed in the supplement.20 (link)
Allprep dna rna purification kit
Manufactured by Qiagen
Sourced in Germany
The Allprep DNA/RNA purification kit is a laboratory tool designed to simultaneously extract and purify high-quality genomic DNA and total RNA from a single biological sample. The kit utilizes a specialized procedure to ensure the efficient recovery of both DNA and RNA molecules from a variety of sample types.
Automatically generated - may contain errors
Lab products found in correlation
Trusight myeloid sequencing panel, by Illumina (2 mentions)
Hiseq rapid sbs kit v2, by Illumina (2 mentions)
Miseq sequencer, by Illumina (1 mentions)
Dneasy blood and tissue kit, by Qiagen (1 mentions)
Miseq reagent kit v3, by Illumina (1 mentions)
Trusight myeloid panel, by Illumina (1 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (1 mentions)
Superscript 4 cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Rdd buffer, by Qiagen (1 mentions)
Dnase, by Qiagen (1 mentions)
Sensimix 2 probe mastermix, by Meridian Bioscience (1 mentions)
Tissuelyzer 2, by Qiagen (1 mentions)
Hiseq 2500 platform, by Illumina (1 mentions)
Genechip human genome u133 plus 2.0 array, by Thermo Fisher Scientific (1 mentions)
Truseq stranded total rna library prep kit, by Illumina (1 mentions)
Kapa htp library preparation kit, by Agilent Technologies (1 mentions)
Sureselectxt clinical research exome kit, by Agilent Technologies (1 mentions)
Oncoscan, by Thermo Fisher Scientific (1 mentions)
Hiseq 2500 sequencer, by Illumina (1 mentions)
9 protocols using allprep dna rna purification kit
1
Genomic Profiling of Myeloid Leukemia
2
Comprehensive Genetic Profiling of Myeloid Leukemia
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Peripheral blood or BM samples from diagnosis were studied centrally by G- and R-banding analysis. Chromosomal abnormalities were described according to the International System for Human Cytogenetic Nomenclature [20 ].
DNA from ficoll-separated BM or PB samples was extracted using the Allprep DNA/RNA purification kit (Qiagen, Hilden, Germany). DNA from whole blood cell pellets was extracted using the DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany). The peqGOLD Micro Spin Tissue DNA Kit was used to extract DNA from cells taken from BM smears (VWR International bvba, Leuven, Belgium). DNA libraries were prepared from diagnostic DNA samples with a custom TruSight Myeloid Panel (Illumina, San Diego, CA, USA) covering genes or gene hotspots of 46 genes recurrently found in myeloid leukemia as previously described [14 (link),15 (link),21 (link)] or with a Custom enrichment panel (Nextera Flex for enrichment, lllumina) covering 48 genes (Supplementary Table S1 ) according to the manufacturers’ instructions. The Illumina Miseq reagent kit v3 (600 cycles) was used for sequencing and was run on the MiSeq sequencer (Illumina).
DNA from ficoll-separated BM or PB samples was extracted using the Allprep DNA/RNA purification kit (Qiagen, Hilden, Germany). DNA from whole blood cell pellets was extracted using the DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany). The peqGOLD Micro Spin Tissue DNA Kit was used to extract DNA from cells taken from BM smears (VWR International bvba, Leuven, Belgium). DNA libraries were prepared from diagnostic DNA samples with a custom TruSight Myeloid Panel (Illumina, San Diego, CA, USA) covering genes or gene hotspots of 46 genes recurrently found in myeloid leukemia as previously described [14 (link),15 (link),21 (link)] or with a Custom enrichment panel (Nextera Flex for enrichment, lllumina) covering 48 genes (
3
Quantitative Analysis of DNA Methylation
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DNA methylation analysis was performed as described previously [22 (link)]. Briefly, genomic DNA samples were purified from eutopic endometrium, ectopic lesions, and the cultured endometrial stromal cells that were transfected with siRNA oligos following the protocol of Allprep DNA/RNA purification kit (Qiagen), then digested with methylation-sensitive, methylation-dependent, both, and non, respectively. Equal amounts of the digested DNAs were then subjected to qRT-PCR following the EpiTect Methyl II Assay protocol (Qiagen) using specific primers flanking CpG islands in the bidirectional promoter (Table 1 ). The methylated or nonmethylated human genomic DNA (New England Biolabs) served as positive or negative controls. Levels of DNA methylation were determined by the average ΔCt values obtained from qPCR amplification of mock-digested, methylation-sensitive enzyme-digested, methylation-dependent enzyme-digested and double-digested DNA samples following product protocol. The fold induction of DNA methylation in the ectopic lesions compared with that in the normal endometrium was determined. The representative samples were submitted to bisulfite sequencing using the primers specific to the promoter region to validate the methylation results obtained from quantitative real-time PCR.
4
Quantifying HEV RNA from Mouse Tissues
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Total RNA from mouse tissues was isolated using the Qiagen AllPrep DNA/RNA purification kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. In brief, 10–20 mg of each tissue was homogenized as previously described [47 (link)] in RLT buffer supplemented with 1% β-mercaptoethanol. After RNA isolation, DNA digestion was performed by incubating 350 ng of RNA per sample with 2.5% DNase and 10% RDD buffer (QIAGEN, Hilden, Germany) for 30 min at room temperature. Reverse transcription was performed using the SuperScript IV cDNA synthesis kit (Thermo Fisher Scientific, Waltham, MA, USA). Gene expression was quantified using the SensiMix™ II Probe Master mix (Bioline, London, UK) on a StepOnePlus™ Real-Time PCR System (Applied Biosystems™, Thermo Fisher Scientific, Waltham, MA, USA) with primers, as listed in Supplemental Table S1 .
The qPCR results were expressed as mean HEV-ORF3 vector genome copy number per µg total RNA (vg/µg). Known copy numbers of the HEV p6 plasmid were serially diluted and used to generate a standard curve.
The qPCR results were expressed as mean HEV-ORF3 vector genome copy number per µg total RNA (vg/µg). Known copy numbers of the HEV p6 plasmid were serially diluted and used to generate a standard curve.
5
High-Quality RNA Extraction and Sequencing
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Adipose biopsy procedures were previously described36 (link). DNA and RNA were extracted from 200 mg of frozen visceral (omental) WAT homogenized using a TissueLyzer-II (QIAGEN, Hilden, Germany) with the AllPrep DNA/RNA purification kit (QIAGEN). High-quality RNA samples (RIN > 8) were used for library construction using the TruSeq Rybo-zero method and were sequenced by the Massively Parallel Sequencing Shared Resource (MPSSR) at OHSU using the Illumina HiSeq. 2500 platform, with the 100-bp, single-read protocol. RNA-seq summary statistics is shown in Supplementary Table S13 .
6
DNA Methylation Profiling of Endometrial Lesions
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Genomic DNA samples were purified from the ectopic lesions and donor’s endometrium following the protocol of Allprep DNA/RNA purification kit (Qiagen), then digested with Methylation-sensitive, Methylation-dependent, or both enzymes, respectively. Equal amounts of digested DNAs were then subjected to qPCR following the EpiTect Methyl II Assay protocol (Qiagen) using specific primers flanking the known/predicted “CG” islands in the promoter regions of mouse Pgr (EPMM111296-1A) and Hoxa10 (EPMM109276-1A), respectively. The methylated or non-methylated lymph node genomic DNA (New England Biolabs) served as positive or negative controls. Levels of DNA methylation were determined by the average ΔCt values obtained from qPCR amplification of mock-digested, methylation-sensitive enzyme-digested, methylation-dependent enzyme-digested and double-digested DNA samples following product protocol. The fold induction of DNA methylation in the ectopic lesions (D16) compared to that in the donor’s endometrium (D0) was determined.
7
Comprehensive Molecular Profiling of Tumors
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DNA and RNA were extracted from the tumor samples stored in RNAlater using Qiagen's AllPrep DNA/RNA purification kit and QIAcube workstation. DNA from whole blood samples was isolated using Tecan's liquid handling automated station. Detailed method descriptions can be found in the Supplementary Material. Briefly, WES was performed on genomic and tumor DNA using Roche's KAPA HTP Library Preparation Kit and Agilent's SureSelectXT Clinical Research Exome kit. Paired-end sequencing was performed on Illumina instruments, with an average coverage of 50–100×. Data were processed using Qiagen's Biomedical Genomics Workbench and Ingenuity Variant Analysis. RNA sequencing was performed using Illumina's TruSeq Stranded Total RNA Library Prep Kit and paired-end sequencing was performed to gain an average output of 50–100 M reads. FusionMap was used for the screening of fusion transcripts [11 (link)], and if positive, validation was carried out by Sanger sequencing. Sequencing data can be found in ENA under accession number PRJEB23819 . Somatic CNAs were detected by SNP arrays (CytoScan or OncoScan; Affymetrix, GSE108089). Data were analyzed using NEXUS (BioDiscovery). Expression levels were analyzed using Affymetrix's GeneChip® Human Genome U133 Plus 2.0 Array, with subsequent processing by Qlucore (Figure 1B ).
8
Cytogenetic and Genomic Profiling of Leukemia
9
Retrospective Review of Mucoepidermoid Carcinoma
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