Genomic DNA (gDNA) and plasma samples were obtained from the PrecisionLink Biobank at Boston Children’s Hospital (BCH). Participants are given the opportunity to also consent to collection of a 4 mL blood sample for research use, from which DNA and plasma aliquots are obtained. In conjunction with other scheduled clinical laboratories, the whole blood is collected from participants in EDTA treated tubes. When received in the Biobank Core Lab, the blood is centrifuged at 2000 × g for 10 min at room temperature. Plasma is then aliquoted into 0.5 mL microcentrifuge tubes and stored at − 80 °C in the Biobank Core Lab facility until requested. gDNA is extracted from the whole blood using Gentra Puregene Extraction Kit (Qiagen Sciences Inc, Germantown, MD) or Chemagic B5k Extraction Kits (PerkinElmer, Waltham, MA) resulting in two 0.225 mL aliquots. DNA samples are stored at − 80 °C until requested for research use at which point they undergo normalization and QC. The PrecisionLink Biobank initiative is approved by the BCH Institutional Review Board (protocol number—P00000159).
Gentra puregene extraction kit
Manufactured by Qiagen
Sourced in Germany
The Gentra Puregene extraction kit is a laboratory product designed for the purification of DNA from a variety of sample types. The kit utilizes a simple and efficient protocol to isolate high-quality genomic DNA from cells or tissues.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (3 mentions)
Nanodrop assay, by Thermo Fisher Scientific (2 mentions)
Picogreen, by Thermo Fisher Scientific (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Sequel 2, by Pacific Biosciences (1 mentions)
Femto pulse, by Agilent Technologies (1 mentions)
Smrtbell express template prep kit 2, by Pacific Biosciences (1 mentions)
G tube, by Covaris (1 mentions)
Gelred nucleic acid stain, by Biotium (1 mentions)
Abi 377 sequencer, by Thermo Fisher Scientific (1 mentions)
Vtec rpla, by Denka Seiken (1 mentions)
Stec plates, by CHROMagar (1 mentions)
Abi 3730 dna analyser, by Thermo Fisher Scientific (1 mentions)
Genemapper 4, by Thermo Fisher Scientific (1 mentions)
12 protocols using gentra puregene extraction kit
1
Biobank Sampling and Storage Protocol
2
Genetic Variant Analysis of VWF and F8
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EDTA-anticoagulated whole blood was obtained from all family members, and genomic DNA was isolated from leukocytes using the Gentra Puregene extraction kit (Qiagen, Germantown, MD, USA). The entire coding region of VWF and F8 of the index case (IC) was sequenced by Sanger methodologies and intron/exon boundaries, the 5′ and 3′ untranslated regions, and the proximal promoter regions of F8. The presence of the novel variant was confirmed via bi-directional sequencing in the IC. Direct sequencing of VWF exon 25 was carried out to detect the variant in the additional family members. Sequencing data can be found in GenBank (ncbi.nlm.nih.gov/genbank, accession numbers OM169364, OM169365, and OM169366).
3
Genomic DNA Extraction from Nobuto Strips
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Total DNA was extracted using a Gentra PureGene extraction kit following the manufacturer's protocol with the following minor modifications (Qiagen Puregene, Qiagen, Hilden, Germany). Peripheral blood on Nobuto strips was incubated for 30-min in 300uL of red blood cell lysis solution. The Nobuto strip was then incubated for 24 h in 600uL of PureGene cell lysis solution at 56 °C to lyse the white blood cells bound to the Nobuto strip. Finally, 10uL of Proteinase K (ProtK) (20mg/uL) was added to each sample containing cell lysis solution and Nobuto strip and incubated at 56 °C for 24 h (Themo Fisher Scientific, Waltham, MA). Total DNA was washed and isolated following the manufacturer's recommended protocol (Qiagen Puregene, Qiagen, Hilden, Germany). Pelleted DNA was resuspended in 50uL of DNA hydration solution (TE buffer) and at 4 °C stored short term until amplification.
4
OXTR Promoter DNA Methylation
5
NKG2C Genotyping from Blood Cells
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Genotyping was assessed on DNA from peripheral blood granulocytes (N=105), peripheral blood mononuclear cells (PBMCs) (N=9), CD3 + (N=4) or CD19 + (N=17) lymphocytes. DNA was extracted using a Gentra Puregene extraction kit (QIAGEN, Hilden, Germany). NKG2C zygosity was assessed as previously described 29 (link) and detailed in Supplemental Methods.
6
High-Molecular-Weight DNA Extraction and HiFi Sequencing
7
Comparative Genomics of Texas Freshwater Mussels
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We sampled P. amphichaenus and P. streckersoni from focal drainages in Texas (i.e., Brazos, Neches, Sabine, and Trinity). Potamilus amphichaenus was collected from 24 localities (Neches = 8, Sabine = 8, Trinity = 8) and P. streckersoni was collected from 22 localities in the Brazos River drainage (Table 1 ). Outgroups were not included considering multiple phylogenetic studies have resolved P. amphichaenus and P. streckersoni as sister species with strong support (Smith et al., 2019 (link), 2020 (link)). Genomic DNA was extracted from fresh mantle clips using the Gentra PureGene extraction kit following manufacturer protocol (Qiagen; Hilden, Germany). High molecular weight DNA was ensured by visualizing isolations on a 1% agarose gel stained with GelRed Nucleic Acid Stain (Biotium), and the purity of each isolation was quantified using a NanoDrop™ (Thermo Fisher Scientific).
8
Loss of Heterozygosity Analysis of BAP1 in Tumor Samples
9
COMT Genotyping in Human Genomic Studies
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Genomic DNA was isolated from ethylenediaminetetraacetic acid (EDTA)-treated whole blood using the Gentra Puregene extraction kit (Qiagen Sciences, Germantown, MD), quantified, and quality checked with Picogreen (Invitrogen, Eugene, OR) and Nanodrop assays (Thermo Scientific, Wilmington, DE). A sequence validated TaqMan (Applied Biosystems) assay was utilized for genotyping which was done blind to behavioral data. We compared G/G homozygotes (i.e., individuals homozygous for the Val allele) to A/A homozygotes and A/G heterozygotes (i.e., carriers of the Met allele). Examining genotype in an additive fashion indicated that A/A homozygotes and heterozygotes displayed nearly identical patterns of results for the PCET, and both groups differed from the G/G homozygotes. Therefore, we collapsed across A-carriers and A/A homozygotes to increase statistical power. We did not observe any deviations from Hardy-Weinberg equilibrium for the COMT rs4680 SNP in our study sample (p = .38).
10
Characterization of E. coli Isolates from Beef Cattle
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Bacterial isolates: A total of 96 rectal samples (one sample from one animal) of Japanese beef cattle raised on 13 farms were acquired from the Osaka Municipal Slaughter Center from August to November 2011. The samples were directly spread on CHROMagar STEC plates (CHROMagar Microbiology, Paris, France), and 3-10 mauve colonies of each sample were selected. The identification of E. coli species and stx-positive strains was confirmed by biochemical tests (utilization of sucrose, lactose, and glucose, production of gas, H2S and indole on TSI and SIM media [Nissui, Tokyo, Japan]; and secretion of lysine decarboxylase on lysine decarboxylase media [self-made]) and a verotoxin gene PCR screening set (Takara Bio Inc., Kusatsu, Japan). Shiga toxin production was also confirmed by a reverse passive latex agglutination (RPLA) assay using VTEC-RPLA (Denka-Seiken, Tokyo, Japan). O-and H-serotypes were determined by means of commercially available antisera (Denka Seiken; 50 O-serotypes and 22 H-serotypes). For broad screening of the O-serotype, all antisera (O1-O187, Serum State Institute, Copenhagen, Denmark) and a PCR-based O-typing method (E. coli O-genotyping PCR) (23) were also used.
DNA extraction: Genomic DNA was extracted using the Gentra PureGene Extraction Kit (Qiagen, Hilden, Germany).
DNA extraction: Genomic DNA was extracted using the Gentra PureGene Extraction Kit (Qiagen, Hilden, Germany).
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