Based on the defined criteria (see above) selected species were subjected to subsequent qPCR analysis. DNA from single individuals was extracted using the DNeasy® 96 Blood & Tissue Kit (Qiagen, Hilden, Germany). Unless otherwise noted, all buffers, racks, and tubes mentioned below are part of this extraction kit. The extraction procedure was a combination of the QIAGEN Supplementary Protocol DY14 (“Purification of total DNA from insects using the DNeasy® Blood & Tissue Kit”; August 2006) adapted to the DNeasy® 96 Blood & Tissue Kit as follows: For DNA extraction, 180 µL phosphate buffered saline (PBS, pH 7.2; 50 mM potassium phosphate, 150 mM NaCl; not part of the DNeasy® Blood & Tissue Kit) and a 3-mm diameter tungsten bead (Qiagen, Hilden, Germany; not part of the DNeasy® Blood & Tissue Kit) were added to every collection microtube containing an insect specimen. The samples were disrupted for 3 min at 30 Hz in the Tissue Lyser II (Qiagen, Hilden, Germany), and the lysates were collected by a short spin centrifugation of the 96-well microcentrifuge plate. Then 20 µL Proteinase K and 200 µL buffer AL (without ethanol) were added, vigorously mixed, and incubated for 10 min at 56 °C, and 200 µL 96% (v/v) ethanol (not part of the DNeasy® Blood & Tissue Kit) was added to each sample and processed following the instruction of the manufacturer.
Dneasy 96 blood tissue kit
Manufactured by Qiagen
Sourced in Germany, China, United Kingdom, United States
The DNeasy 96 Blood & Tissue Kit is a high-throughput DNA extraction solution designed for the purification of genomic DNA from various biological samples, including blood, tissue, and cells. The kit utilizes a silica-based membrane technology to efficiently capture and purify DNA, while removing contaminants and inhibitors. It is suitable for a wide range of downstream applications, such as PCR, sequencing, and genotyping.
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Lab products found in correlation
Nanodrop 1000 spectrophotometer, by Thermo Fisher Scientific (6 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (4 mentions)
Rneasy 96 kit, by Qiagen (3 mentions)
Picogreen, by Thermo Fisher Scientific (2 mentions)
Atl buffer, by Qiagen (2 mentions)
Clonesaver fta filter paper cards, by GE Healthcare (2 mentions)
Harris 3 mm micro puncher, by GE Healthcare (2 mentions)
Rlt buffer, by Qiagen (2 mentions)
Onestep pcr inhibitor removal kit, by Zymo Research (2 mentions)
Allprep dna rna mini kit, by Qiagen (2 mentions)
Proteinase k, by Qiagen (2 mentions)
Abi 3730 genetic analyser, by Thermo Fisher Scientific (2 mentions)
Lightcycler 480, by Roche (2 mentions)
Dneasy blood tissue kit, by Qiagen (2 mentions)
Tissuelyser 2, by Qiagen (2 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Spectramax m3 microplate reader, by Molecular Devices (1 mentions)
Hiseq x instrument, by Illumina (1 mentions)
Truseq dna pcr free protocol, by Illumina (1 mentions)
Hiseq 4000 instrument, by Illumina (1 mentions)
63 protocols using dneasy 96 blood tissue kit
1
DNA Extraction from Insect Specimens
2
Spatial Sampling of Fish Populations
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We collected tissue samples from fish above and below the Union Street Dam for each of our five study species from 2017 to 2019. All sampling was performed with IACUC approval under University of Wisconsin—Stevens Point protocol number 2019.03.05. Fish below the dam were captured by boat electrofishing from the mouth of the Boardman River up to the Union Street Dam. Samples were taken during routine monitoring surveys, with the vast majority of fish being captured between April and July, and sampling spread relatively equally across 2017 and 2018, with only a few samples collected in 2019 (Figure S1 ). Field crews were unable to capture yellow perch in this river section, and we therefore acquired tissue samples from fish captured by ice anglers nearby (~1 km away) in Grand Traverse Bay during February 2018. Above the dam, fish were collected in Boardman Lake using experimental gillnets, mini fyke nets, or boat electrofishing. All above‐dam samples were collected in June 2019 with the exception of 14 walleyes collected in March 2018. Fish were identified to species, measured (mm total length), and tissue samples from the caudal or pelvic fins were collected and preserved in 95% ethanol. DNA was extracted from fin tissue with DNeasy® 96 Blood & Tissue Kits (Qiagen).
3
High-Density Genotyping of Chicken Populations
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Genomic DNA was extracted from 1.5-mL blood samples using DNeasy 96 Blood & Tissue Kits (QIAGEN, Germany). A total of 1078 hens were genotyped with the Chicken 600 K SNP array [42 (link)] (Affymetrix, Inc. Santa Clara, CA, USA) which contained 580,961 SNPs across 28 autosomes and two sex chromosomes. We first discarded 6593 SNPs with unknown physical position and repeated genomic coordinates. The Affymetrix Power Tools v1.19.0 (APT) software was then implemented to control the quality of sample call rate (> 97%) and dish quality (> 0.82). After the quality control, 1063 individuals and 517,856 SNPs remained. In addition, the low quality of SNPs (SNP call rate < 95%, minor allele frequency < 0.01, Hardy-Weinberg equilibrium P < 1 × 10–6) were filtered out through the PLINK package [43 (link)]. The remaining SNPs with missing genotypes were imputed using the Beagle v4.0 procedure [44 (link)]. Finally, a total of 1063 individuals and 294,705 SNPs located on autosomes (Additional file 4 : Table S2) were deemed eligible for the following analyses.
4
Whole Genomic DNA Extraction from Flounder
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To extract whole genomic DNA from adult summer flounder, ≤ 20 mg of muscle tissue or fin was lysed, and DNA was washed and eluted using DNeasy 96 Blood & Tissue Kits (QIAGEN; Hilden, Germany) and the manufacturer's recommended protocols. DNA extracts were visualized on 2% agarose gels to assess quality and were subsequently quantified using PicoGreen (Thermo Fisher Scientific, Waltham, MA, USA) and a SpectraMax M3 Microplate Reader (Molecular Devices; Sunnyvale, CA, USA).
5
Genome Sequencing of Commercial Pigs
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Twenty-six commercial pigs were used in this study. Tissue samples were collected from ear punches or tail clippings and genomic DNA was extracted using Qiagen DNeasy 96 Blood & Tissue kits (Qiagen Ltd., Mississauga, ON, Canada). Paired-end library preparation was conducted using the TruSeq DNA PCR-free protocol (Illumina, San Diego, CA). Two sets of libraries were produced; one with an average insert size of 350 bp and the other with an average insert size of 550 bp. Libraries with an average insert size of 350 bp were sequenced on a HiSeq 4000 instrument (Illumina, San Diego, CA), for a target coverage of 2× per sample. For this, all 26 samples were multiplexed within a single flow cell channel. Libraries with an average insert size of 550 bp were sequenced on a HiSeq X instrument (Illumina, San Diego, CA), for a target coverage of 30× per sample. For this, the 26 samples were sequenced, one sample per flow cell channel. All libraries were sequenced at Edinburgh Genomics (Edinburgh Genomics, University of Edinburgh, Edinburgh, UK). DNA samples from the same pigs were also genotyped using the GGP-Porcine HD BeadChip (GeneSeek, Lincoln, NE).
6
Genome-wide SNP genotyping in common carp
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Genomic DNA was extracted from whole blood using a DNeasy 96 Blood & Tissue Kit (Qiagen, Shanghai, China) following the manufacturer’s protocol. The extracted DNA was quantified using a NanoDrop-1000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, United States). The integrity of DNA was examined on a 1.5% agarose gel by electrophoresis. The final DNA concentration was diluted to 50 ng/μL for genotyping. The total amount of qualified genomic DNA for whole genome sequencing was 2 μg per sample. The common carp 250K SNP array was developed in a previous study using the Affymetrix Axiom genotyping technology (Xu J. et al., 2014 (link)). Genotyping was performed by GeneSeek (Lincoln, Nebraska, United States). After genotyping, PLINK v1.93 was used for quality control (Chang et al., 2015 (link)). SNPs with low call rate (<95%) or low minor allele frequency (MAF < 5%) were excluded, and samples with <90% genotyping rates were filtered out.
7
DNA Extraction from Mosquitoes and Dried Blood Spots
8
Quantitative Analysis of Transient Wolbachia Infection
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Wolbachia density and distribution in the transient infected mosquitoes were compared 7 and 14 days post injection (dpi) to the wAlbB line using qPCR and fluorescence in situ hybridisation (FISH). DNA was extracted from stable and transiently Wolbachia infected mosquitoes using the DNeasy 96 Blood & Tissue kit (Qiagen) according to the manufacturer’s specifications. Quantitative PCR to determine the total relative Wolbachia densities of infected lines was performed as described by [30 (link)] using primers specific to the gene coding for the Wolbachia surface protein (wsp) (forward primer 5’-GCATTTGGTTAYAAAATGGACGA-3’, reverse primer 5’- GGAGTGATAGGCATATCTTCAAT-3’), as well as the Ae. aegypti actin gene (forward primer 5’- GACTACCTGATGAAGATCCTGAC-3’, reverse primer: 5’- GCACAGCTTCTCCTTAATGTCAC-3’) [24 (link)]. Statistical differences were determined using a Mann-Whitney (Graphpad Prism version 6.0f).
Wolbachia was localized in sections of paraffin-embedded 5–7 day old female mosquitoes by FISH, as described in [31 (link)], except that only one probe against 16S rRNA was used and its concentration was increased 10-fold to improve the signal. wAlbB was detected using AlbBW5: 5’-CTTAGGCTTGCGCACCTTGCAA-3’, labelled with Alexa 488 dye (green). DAPI was used to stain total DNA.
Wolbachia was localized in sections of paraffin-embedded 5–7 day old female mosquitoes by FISH, as described in [31 (link)], except that only one probe against 16S rRNA was used and its concentration was increased 10-fold to improve the signal. wAlbB was detected using AlbBW5: 5’-CTTAGGCTTGCGCACCTTGCAA-3’, labelled with Alexa 488 dye (green). DAPI was used to stain total DNA.
9
qRT-PCR Analysis of Gene Expression and Mitochondrial DNA Copy Number
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cDNA templates were synthesized with the cDNA synthesis kit (Applied Biological Materials Inc. (Abm), #G236) using random primers. The cDNA was then subjected to PCR amplification using 2X PCR Taq MasterMix (Abm) and gene-specific primers. Quantitative real-time PCR analysis was performed with PowerUP SYBR Green Master Mix (Applied Biosystems) reagent. Reactions were performed in triplicate. Relative gene expression was calculated using the comparative Ct (2−∆∆Ct) method81 (link), where values were normalized to a 5S or 18S rRNA gene expression. The mRNA expression of candidate genes was determined by QuantStudio 6 Flex Real-Time PCR (Life technologies). All primer sequences for qRT-PCR are provided in Supplementary Table 1 .
Mitochondrial DNA (mtDNA) copy number was measured by qRT-PCR. Total genomic DNA was isolated from BAT tissues using a DNeasy 96 Blood & Tissue Kit (QIAGEN, #69504). Then, 100 ng total DNA was used for mtDNA quantification. The copy number was normalized to nuclear DNA. The primer sequences are provided in Supplementary Table1 .
Mitochondrial DNA (mtDNA) copy number was measured by qRT-PCR. Total genomic DNA was isolated from BAT tissues using a DNeasy 96 Blood & Tissue Kit (QIAGEN, #69504). Then, 100 ng total DNA was used for mtDNA quantification. The copy number was normalized to nuclear DNA. The primer sequences are provided in Supplementary Table
10
DNA Extraction from S. pneumoniae and Whole Blood
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DNA extraction from S. pneumoniae isolates was carried out by alkaline lysis as described previously [16 (link)]. For DNA extraction from whole blood, initial pretreatment of the samples was performed as described elsewhere [17 (link)]. Briefly, 100 μL of whole-blood EDTA was added to 100 μL of Tris-EDTA buffer containing 75 U/mL of mutanolysin (Sigma Chemical Co., St. Louis, MO), and the mixture was incubated for 1 hour at 37°C. DNA extraction was then carried out with the DNeasy 96 Blood & Tissue Kit (Qiagen, Venlo, the Netherlands), according to the manufacturer’s instructions.
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