The Qiagen AllPrep DNA Mini Kit, Qiagen, Valencia, CA, was used to isolate nucleic acids from the COLO829 and COLO829BL cell pellets. Specifically, 600 μl of Buffer RLT plus was added to the thawed pellets to disrupt the cells. The lysates were transferred to QiaShredder columns, Qiagen, for homogenization. Genomic DNA purification was conducted as directed by the AllPrep DNA/RNA Mini Handbook, Qiagen. DNA was quantified using the Qubit 2.0 Fluorometer (Life Technologies) and the Nanodrop spectrophotometer (Thermo Scientific; Waltham, MA), and 260/280 and 260/230 absorbance ratios were evaluated for purity. DNA was also electrophoretically separated on a 1% TAE gel to verify the presence of high molecular weight DNA.
Allprep dna mini kit
Manufactured by Qiagen
Sourced in Germany, United States
The AllPrep DNA Mini Kit is a laboratory equipment product designed for the simultaneous purification of genomic DNA, total RNA, and miRNA from a single sample. It utilizes a spin-column-based nucleic acid extraction method to provide high-quality nucleic acid yields from a variety of sample types.
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Lab products found in correlation
Dna blood mini kit, by Qiagen (3 mentions)
7900ht fast real time pcr system, by Thermo Fisher Scientific (3 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (2 mentions)
Hiseq 2500, by Illumina (2 mentions)
Karyostudio v1, by Illumina (2 mentions)
Genomestudio, by Illumina (2 mentions)
Human cytosnp 12v 21 array, by Illumina (2 mentions)
Qubit dna hs assay kit, by Thermo Fisher Scientific (2 mentions)
Qubit dna broad range assay kit, by Thermo Fisher Scientific (2 mentions)
Tapestation system, by Agilent Technologies (2 mentions)
Genomic dna screentape, by Agilent Technologies (2 mentions)
Red blood cell lysis buffer, by BioLegend (2 mentions)
Magmax cell free total nucleic acid isolation kit, by Thermo Fisher Scientific (2 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Qiashredder column, by Qiagen (1 mentions)
Allprep dna rna mini handbook, by Qiagen (1 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (1 mentions)
Sureselect human all exon v6 kit, by Agilent Technologies (1 mentions)
Hiseq 4000 system, by Illumina (1 mentions)
Qiaamp dna blood mini kit, by Qiagen (1 mentions)
30 protocols using allprep dna mini kit
1
COLO829 DNA Extraction Using Qiagen Kit
2
Tissue Sample Preparation and DNA Extraction
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All tumour samples and normal samples were snap frozen. Fresh-frozen samples were divided into three segments and a piece of tissue from the top and bottom part of each segment was sectioned, stained with haematoxylin and eosin and extensive pathological review was carried out by a trained pathologist to ensure there was a minimum of 30% tumour within the segment of the tumour samples and no tumour present in the normal samples. DNA was extracted from the tumour and normal samples using an AllPrep DNA mini kit (Qiagen, Hilden, Germany), and from whole blood samples using a DNA blood mini kit (Qiagen), according to the manufacturers protocol. DNA was quantified by Qubit fluorometer (Invitrogen, Carlsbad, CA, USA) and DNA integrity was examined by agarose gel electrophoresis.
3
Whole-Exome Sequencing of Pre-neoCRT Biopsies
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Four pre-neoCRT biopsies left after scRNA-seq were used for bulk whole exome sequencing. Tissue and blood genomic DNA was extracted using the AllPrep DNA Mini Kit (QIAGEN, Germantown, MD) and QIAamp DNA blood mini kit (QIAGEN), respectively, and then randomly broken into fragments with a length of 180–280 bp. The library construction and capture were performed with the SureSelect Human All Exon V6 kit (Agilent, Santa Clara, CA) according to the manufactures’ protocol. Qualified captured libraries were sequenced at 150 paired-end cycles on a HiSeq4000 system (Illumina). Raw fastq data were quality-trimmed, and adapter sequences were removed, and aligned to the human reference genome (human_g1k_v37) using Burrows-Wheeler Aligner (v0.7.8-r455). Samblaster (v0.1.21)22 (link) was used to select split reads and discord reads from the initial comparison results. Duplicate reads were marked with sambamba (v0.6.3).23 (link) FastQC (v0.11.5) (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/ ) and Qualimap (v2.2)24 (link) were used to perform quality checks on the fastq and bam files, respectively. Tissue copy number variation analysis calls were made by Controlfreec (v11.6)25 (link) with matched blood genomic as normal control. Significant regions of copy number alteration were selected using default parameters at a q value threshold of 0.25 with GISTIC2 (v2.0.23).26 (link)
4
Fibroblast DNA Isolation and Exome Sequencing
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DNA from fibroblast cell lines was isolated from whole-cell lysates using the AllPrep DNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. Exonic regions were enriched using the SureSelect Human All Exon kit from Agilent (Supplementary Data 3 ) followed by sequencing as 100 bp paired-end runs on an Illumina HiSeq2000 and Illumina HiSeq2500 (AG_50MB_v4 and AG_50MB_v5 exome kit samples) or as 76 bp paired-end runs on the Illumina GAIIx (AG_38MB_v1 and AG_50MB_v3 exome kit samples)54 (link).
5
Genetic Biomarker Assessment for Sepsis
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DNA extraction and SNP genotyping was performed according to the manufacturer’s instructions in the laboratories and under the supervision of the Department of Clinical Pharmacology, University Medical Center Goettingen, Germany.
Whole blood samples were drawn from all study subjects within 72 h after sepsis onset. The extraction of genomic DNA was performed using either the QIAmp® DNA Blood Kit in QIAcube®, the EZ1® DNA Blood Kit in BioRobot EZ1® or the AllPrep DNA Mini Kit (all from Qiagen, Hilden, Germany), as previously described [21 (link),24 (link),43 (link)]. Quantity and quality of the extracted DNA were tested by spectrophotometric measurement.
The LAG-3 rs951818 was genotyped in all samples through TaqMan polymerase chain reaction (PCR) using the appropriate predesigned TaqMan® SNP Genotyping Assay C___8921385_10 (Thermo Fisher Scientific, Waltham, MA, USA) and a 7900HT Fast-Real-Time PCR System (Life Technologies, Darmstadt, Germany) as well as 7900HT Fast-Real-Time PCR System software (SDS v2.4.1 for Windows 7, Applied Biosystems, Foster City, CA, USA). Over 20% of the samples were genotyped in duplicate to increase reliability.
Whole blood samples were drawn from all study subjects within 72 h after sepsis onset. The extraction of genomic DNA was performed using either the QIAmp® DNA Blood Kit in QIAcube®, the EZ1® DNA Blood Kit in BioRobot EZ1® or the AllPrep DNA Mini Kit (all from Qiagen, Hilden, Germany), as previously described [21 (link),24 (link),43 (link)]. Quantity and quality of the extracted DNA were tested by spectrophotometric measurement.
The LAG-3 rs951818 was genotyped in all samples through TaqMan polymerase chain reaction (PCR) using the appropriate predesigned TaqMan® SNP Genotyping Assay C___8921385_10 (Thermo Fisher Scientific, Waltham, MA, USA) and a 7900HT Fast-Real-Time PCR System (Life Technologies, Darmstadt, Germany) as well as 7900HT Fast-Real-Time PCR System software (SDS v2.4.1 for Windows 7, Applied Biosystems, Foster City, CA, USA). Over 20% of the samples were genotyped in duplicate to increase reliability.
6
CTLA-4 Genotyping from Blood Samples
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DNA was automatically extracted from 200 µl of EDTA blood using a QIAamp® DNA Blood Kit in a QIAcube® or from 350 µl of EDTA blood using an EZ1® DNA Blood Kit in a BioRobot EZ1® or from peripheral blood mononuclear cells (PBMCs) using an AllPrep DNA Mini Kit according to the manufacturer’s instructions (all from Qiagen, Hilden, Germany). The quantity and quality of the DNA were determined by spectrophotometric measurement and revealed an average concentration of 33.8 ng/µl and an average E260/E280 extinction ratio of 1.85. Genotyping of the extracted DNA was performed via TaqMan polymerase chain reaction (PCR) using the predesigned TaqMan® SNP Genotyping Assay C___2415786_20 and a 7900HT Fast Real-Time PCR System (TaqMan) according to the manufacturer’s instructions (Life Technologies, Darmstadt, Germany). The genotyping outcomes were generated using 7900HT Fast Real-Time PCR System Software (SDS v2.4.1 for Windows 7, Applied Biosystems, Foster City, USA). For reliability reasons, 20% of the samples were genotyped in duplicate and showed completely concordant results. The DNA extraction and CTLA-4 rs231775 genotyping were performed entirely in the laboratories and under the supervision of the Department of Clinical Pharmacology of the University Medical Center Goettingen.
7
Bisulfite Sequencing of CTLA-4 Promoter
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Bisulfite sequencing was performed as described previously [40 (link)]. Genomic DNA was prepared using an AllPrep DNA Mini Kit (Qiagen, Hilden, Germany). DNA methylation was detected in T cell subsets at the promoter region of CTLA-4. DNA was bisulfite treated using an EpiTect Plus Bisulfite Conversion Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. PCR products were purified and sequenced. DNA methylation analysis was carried out using quantification tool for methylation analysis, and methylation was determined at each CpG dinucleotide [41 (link)].
8
Genotyping of Peripheral Blood Monocytes
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Peripheral blood monocytes (PBMCs) from approximately 30 ml of heparinized peripheral blood were isolated through Ficoll density gradient centrifugation according to standard procedures described previously
[33 (link)]. Cell preparations were routinely assessed for viability (>95%) by trypan blue dye exclusion.
Genomic DNA (gDNA) was purified from PBMCs using the AllPrep DNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. The isolated nucleic acid concentration and purity were determined by 260 and 280 nm optical density readings. DNA integrity was evaluated through 0.6% agarose gel electrophoresis.
Genotyping was performed using 4 ng of PBMC-derived gDNA in a commercially available genotyping assay (Assay ID C_31784034_10, Applied Biosystems, Darmstadt, Germany) in a total volume of 10 μl. The reactions were performed in a StepOnePlus sequence detection system (Applied Biosystems, Darmstadt, Germany) according to the supplier’s instructions.
[33 (link)]. Cell preparations were routinely assessed for viability (>95%) by trypan blue dye exclusion.
Genomic DNA (gDNA) was purified from PBMCs using the AllPrep DNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. The isolated nucleic acid concentration and purity were determined by 260 and 280 nm optical density readings. DNA integrity was evaluated through 0.6% agarose gel electrophoresis.
Genotyping was performed using 4 ng of PBMC-derived gDNA in a commercially available genotyping assay (Assay ID C_31784034_10, Applied Biosystems, Darmstadt, Germany) in a total volume of 10 μl. The reactions were performed in a StepOnePlus sequence detection system (Applied Biosystems, Darmstadt, Germany) according to the supplier’s instructions.
9
DNA Extraction and Bisulfite Conversion
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DNA was extracted from the frozen tissue and urine samples using the AllPrep DNA Mini kit (Qiagen, Inc., Valencia, CA, USA) according to the manufacturer's protocol. The concentration and purity of DNA were assessed by determining the OD260/OD280 ratio using a Nanodrop 1000 spectrophotometer (Thermo Fisher Scientific, Inc., Waltham, MA, USA). DNA was treated by bisulfite using an EZ-96 DNA Methylation-Gold™ kit (Zymo Research, Irvine, CA, USA) according to the manufacturer's protocol, as well as the previously described protocol (20 (link)), and stored at −80°C until use.
10
Chorionic Villi DNA Extraction and SNP Analysis
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The chorionic villi were thoroughly separated from the maternal deciduas as previously published to avoid maternal genome contamination [35 (link)]. Total DNA from the chorionic villi was extracted using the All Prep DNA Mini Kit (Qiagen, Germany) and subjected to SNP array using the Human CytoSNP-12v.21 Array (Illumina, San Diego, California, USA). The SNP array data was analyzed using Genome-Studio (Illumina 2011) and Karyo-Studio v1.4 (Illumina 2011). The copy number variants (CNVs) were mapped using the DGV database (http://dgv.tcag.ca/dgv/app/faq ) to identify the candidate pathogenic CNVs. All the steps were analyzed independently by at least two expert technicians.
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