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16 protocols using picopure dna extraction kit

1

Tumor Cellularity Assessment and DNA Extraction

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Hematoxylin and eosin stained tissue sections of tumor biopsy samples were reviewed by a pathologist who circled the tumor area and estimated tumor cellularity in a circled area. Specimens with a minimum of 20% tumor in the circled area were selected for this study. DNA was extracted from FFPE tumor samples using a PicoPure DNA extraction kit (Arcturus, Mountain View, CA) and purified using the Agencourt AMPure XP kit (Agencourt Biosciences, Beverly, MA). Qubit DNA high-sensitivity assay kit (Life Technologies, Carlsbad, CA) was used to quantify purified DNA.
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2

Quantifying KDR Gene Copy Number

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Histologic sections were reviewed by a lung cancer pathologist to assess presence, quantity, quality, and histologic types of tumor tissues. At least 500 malignant cells in the tissue specimens were required to be considered adequate for analysis for copy number. To enrich for malignant cell content (>70% malignant cells), tumor tissues were microdissected from formalin-fixed, paraffin-embedded (FFPE) tissue sections for subsequent DNA extraction using the Pico Pure DNA Extraction Kit (Arcturus). KDR gene copy number was detected using methods described previously (26 (link)). For qPCR, we utilized the ABI 7300 Real-Time PCR System (Applied Biosystems) along with an endogenous (Actin) control, and all experiments were performed in triplicate. Gene copy number of greater than 4 was considered as CNG. KDR gene copy number was also examined for using FFPE 4 mm histology sections using an assay utilizing a BAC probe containing KDR sequences labeled SpectrumRed and a commercial CEP 15 probe (Abbott Molecular) labeled in SpectrumGreen or similar method. All probes were validated in normal specimens for chromosomal mapping and appropriate specificity and sensitivity with a centromeric 4q probe as an internal control.
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3

Laser Microdissection and Genomic DNA Extraction

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Laser microdissection was performed using a Nikon Eclipse TE 2000-S laser capture microscope system (Nikon Instruments, Tokyo, Japan) as previously reported [70 (link)]. Briefly, 5-μm formalin-fixed tissue sections were deparaffinized and immunohistochemically stained with anti-β-catenin antibody. Intense or weak stained areas were selected, collected by laser capture microdissection, and genomic DNA was extracted using PicoPure DNA extraction kit (Arcturus Bioscience, Mountain View, CA, U.S.A.) in a total volume of 55 μl. Genomic DNA from cell cultures was extracted using Tissue Kit (Qiagen, Milan, Italy) About 3–5 ml of genomic DNA was subject to PCR in a total volume of 50 ml containing 25 ml of 2x PCR Master Mix (Promega, Madison, WI, USA) and 25 pmol of forward primer 5′-GCTGATTTGATGGAGTTGGA-3′ and reverse primer 5′-GCTACTTGTTCTTGAGTGAA-3′. DNA fragments were checked by electrophoresis in 2% agarose gel and purified using High Pure PCR Product Purification Kit (Roche Diagnostics GmbH, Mannheim, Germany) before sequence analysis.
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4

DNA Extraction and NGS-based Mutation Detection

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The Pico Pure DNA Extraction Kit (Arcturus, Mountain View, CA, USA) and the Agentcourt AMPureXP Kit (Agentcourt Biosciences, Beverly, MA, USA) were employed for DNA purification from marked areas of FFPE tumor sections from each specimen (usually five unstained slides with a minimum of 20% tumor cellularity). Approximately 10 ng (50-gene panel) or 20 ng (134-gene panel) of purified DNA were applied for targeted NGS-based DNA-seq for detections of gene mutations and gene amplifications, as reported previously [10 (link),11 (link),29 (link),30 (link)]. DNA sequencing was performed using the Ion Torrent PGM, but the number of target genes was historically expanded from 50 (Ion AmpliSeq Cancer Hotspot Panel, Thermo Fisher Scientific, Waltham, MA, USA) to 134 (OncoMine Comprehensive Assay, Thermo Fisher Scientific), referred to as the “50-gene panel” and “134-gene panel”, respectively [10 (link),11 (link),29 (link),30 (link)]. However, genes such as ALK, BRAF, EGFR, KRAS, RET and many other mutations that are considered to be hotspots for lung cancer are included in both 50-gene and 134-gene panels. DNA extraction from peripheral blood of the same individual (in most patients) was used as a control for the differentiation of constitutional and somatic aberrations in tumor specimens.
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5

Treg-Specific Deletion of CnB

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Deletion of the floxed CnB alleles upon tamoxifen treatment of Foxp3creERT2 × CnBf/f mice was assessed in Treg cells from tdLN and tumors. We took advantage of the expression of GFP from the Foxp3creERT2 (Foxp3GFP-creERT2) allele to FACS-purify CD4+GFP+ Treg cells by double-sorting to achieve > 98% purity. DNA was extracted immediately using the PicoPure DNA extraction kit (Arcturus) and the rearrangement of the CnB locus was analyzed by PCR.
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6

DNA Extraction and Amplification for Lymphoma

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Regarding non-Hodgkin lymphoma cases, the microdissected cells were adhered to a CapSure cap with adhesive transfer film (Arcturus, MWG-Biotech) and then collected into one standard microcentrifuge tube for the DNA extraction by using PicoPure DNA extraction kit Arcturus (Milan, Italy). Regarding FFPE cHL cases from Siena, DNA from 300 to 600 microdissected cells was extracted by the QIAmp DNA Mini Kit (Qiagen Ltd, Crawley, UK) and then subjected to whole-genome amplification (WGA) using the GenomePlex WGA2 kit (Sigma-Aldrich). The amplified DNA (ranging from 2 to 5 mg) was purified using the GenElute PCR clean-up kit (SigmaAldrich) for subsequent PCR amplification. Regarding frozen cHL cases from Perugia, DNA had been extracted and subjected to duplicate WGA as previously described [27 (link)]. Full details are given in the Supplementary Materials.
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7

Illumina Sequencing Sample Preparation

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Sequencing sample was prepared using a protocol from J.S. Weissman’s laboratory (https://weissmanlab.ucsf.edu/CRISPR/IlluminaSequencingSamplePrep.pdf) except that genomic DNA of samples <10,000 cells was extracted with the Arcturus PicoPure DNA Extraction Kit.
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8

Laser Microdissection and DNA Extraction

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Crypts of interest were microdissected using an LMD7000 compound laser microdissection microscope (Leica) and collected in skirted 96 well plates. Crypts were lysed in Proteinase K/Reconstitution buffer using PicoPure® DNA Extraction Kit (Arcturus®). DNA extraction was done at 65 °C for 3 h, followed by 75 °C for 30 min, and kept at −20 °C before library preparation.
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9

Exome Sequencing of Tumor Samples

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Samples from both institutions were processed and analyzed at MD Anderson. For each sample, representative areas of tumor (ETH, n = 25 and MDA, n = 29) and, when available, normal tissue (MDA, n = 13) were selected and circled on a hematoxylin and eosin–stained slide. These were used as a guide for manual microdissection performed on 10 to 20 unstained 5-mm FFPE sections. The genomic DNA was extracted using the PicoPure DNA Extraction Kit (Arcturus) and purified using the AMPureXP Kit (Agencourt Biosciences). For samples for which corresponding normal tissue was not available, a pool of peripheral blood DNA from 11 healthy individuals was used as a normal reference.
Whole exome libraries were created from the extracted DNA using the hybridization capture–based target enrichment sequencing panel SureSelect Human All Exon V4 (Agilent Technologies, Inc.), which targets 51 Mb of the human genome (human genome assembly GRCh37/hg19) covering exonic regions from 20,965 genes, and then sequenced on a HiSeq4000 sequencer (Illumina Inc.).
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10

Laser Capture Microdissection of Colonic Tissue

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To perform laser capture microdissection (LCM) on colonic thin sections, 4 and 3 fecal masses were analyzed for the Fiber-rich (FR) and Fiber-free (FF) groups, respectively. Colonic thin sections that were deposited on microscope slides were deparaffinized in xylene followed by dehydration by isopropanol (see details in the immunofluorescence staining protocol below). The sections were stored overnight in a container with Drierite dessicant (Drierite, USA). LCM was carried out using a Veritas Microdissection instrument (Arcturus, USA). DNA was extracted from the microdissected samples using the Arcturus Pico Pure DNA extraction kit and the accompanying protocol. In order to perform Ilumina sequencing, 16S rRNA genes were amplified from the LCM-derived samples using a low biomass-optimized touch down PCR protocol as follows: denaturation at 95°C for 2 min; a total of 20 cycles with a touch-down program: denaturation at 95°C for 20 s, extension at 72°C for 5 min, annealing starting at 60°C for 15 s which decreased 0.3°C per cycle; a total of 20 cycles: extension at 72°C for 5 min, annealing at 55°C 15 s and extension at 72°C for 5 min; final extension at 72°C for 5 min. Note that a 5 min extension was used in order to reduce chimera development. Library preparation and sequencing were carried out using similar protocols described for fecal and cecal samples (see below).
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