We analyzed 1,446 adult C. frigida, sampled at 16 locations spanning over 10° of latitude (fig. 1A ) in September/October 2016. Sampling, genotyping, and phenotyping are described in detail in Mérot et al. (2018) . Size was estimated using wing length as a proxy for 1,426 flies. Genomic DNA was extracted using a salt extraction protocol (Aljanabi and Martinez 1997 (link)) with an RNase A treatment (Qiagen). A total of 1,438 flies were successfully genotyped at the Cf-Inv(1) inversion using a molecular marker (Mérot et al. 2018 ).
Rnase a treatment
Manufactured by Qiagen
Sourced in Germany, Australia
RNase A treatment is a lab equipment product designed to degrade RNA molecules. It is a widely used tool in molecular biology and biochemistry laboratories.
Automatically generated - may contain errors
Lab products found in correlation
Qiaamp micro kit, by Qiagen (1 mentions)
Infinium methylationepic beadchip assay, by Illumina (1 mentions)
Infinium methylationepic beadchip assay epic array, by Illumina (1 mentions)
Hiscan, by Illumina (1 mentions)
Broad range kit, by Agilent Technologies (1 mentions)
Qubit 3.0 fluorometer, by Agilent Technologies (1 mentions)
Nextseq 500 high output kit 75 cycles, by Illumina (1 mentions)
Nextseq 500 sequencing platform, by Illumina (1 mentions)
Rneasy mini kit 50, by Qiagen (1 mentions)
Trizol ls reagent, by Thermo Fisher Scientific (1 mentions)
Agilent 2100 bioanalyser, by Agilent Technologies (1 mentions)
Dneasy powermax soil kit, by Qiagen (1 mentions)
Quantifluor dsdna sample kit, by Promega (1 mentions)
Rnase free dnase set kit, by Qiagen (1 mentions)
C57bl 6j mice, by Jackson ImmunoResearch (1 mentions)
Dneasy blood and tissue mini kit, by Qiagen (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Rnalater, by Qiagen (1 mentions)
6 protocols using rnase a treatment
1
Latitudinal Cline in C. frigida
2
Genome-wide DNA Methylation Profiling of Human Lung Fibroblasts
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Genomic DNA was extracted from 2 × 105 primary human lung fibroblasts isolated from fresh and cryopreserved tissue from 3 donors in technical replicates using QIAamp Micro Kit (Qiagen) following the manufacturer’s protocol, with an additional RNase A treatment step (Qiagen).
Infinium MethylationEPIC BeadChip assay genome-wide DNA methylation profiles of human lung fibroblasts were generated using Illumina’s Infinium MethylationEPIC Bead Chip assay (EPIC Array) at the Genomics and Proteomics Core Facility at DKFZ (Heidelberg, Germany). The assay allows determination of DNA methylation levels at more than 850,000 CpG sites and provides coverage of CpG islands, RefSeq genes, ENCODE open chromatin, ENCODE transcription factor binding sites, and FANTOM5 enhancers. The assay was performed according to the manufacturer’s instructions and scanned on an Illumina HiScan. To avoid batch effects, both fresh and cryopreserved samples from the same donor were assayed on the same array.
Infinium MethylationEPIC BeadChip assay genome-wide DNA methylation profiles of human lung fibroblasts were generated using Illumina’s Infinium MethylationEPIC Bead Chip assay (EPIC Array) at the Genomics and Proteomics Core Facility at DKFZ (Heidelberg, Germany). The assay allows determination of DNA methylation levels at more than 850,000 CpG sites and provides coverage of CpG islands, RefSeq genes, ENCODE open chromatin, ENCODE transcription factor binding sites, and FANTOM5 enhancers. The assay was performed according to the manufacturer’s instructions and scanned on an Illumina HiScan. To avoid batch effects, both fresh and cryopreserved samples from the same donor were assayed on the same array.
3
Milk DNA Extraction and Purification
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The enriched raw milk (1 ml) was centrifuged and the raw milk fat layer was removed before the pellet was washed with 1× PBS as described above for raw milk screening. DNA extraction and purification was performed using the MasterPure complete DNA and RNA extraction and purification kit (Lucigen), following the manufacturer’s instructions and including an RNase A treatment for 30 min (Qiagen; 100 mg ml−1) [26 (link)]. Genomic DNA was quantified using a Qubit 3.0 fluorometer and the broad range kit (Agilent), and quality control of the DNA extracts was assessed using the Nanodrop 1.0 or 2.0 spectrophotometer (Agilent).
4
Profiling sEV-Encapsulated RNA via NGS
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sEV RNA was extracted using the RNeasy Mini Kit 50 (Qiagen, Australia) and TRIzol LS Reagent (Life Technologies, Australia). RNaseA treatment (100 ng/mL, Qiagen, Australia, 37 °C for 10 min) was used to select only RNA encapsulated within sEVs as previously described [23 (link)]. The total RNA yield (comprising of mostly small RNA), composition and quality was analyzed using the Agilent 2,100 Bioanalyser for small RNA profiles. Sequencing libraries were generated using the TruSeq® SmallRNA Library Prep Kit, according to the manufacturer’s instructions and as we previously described [24 ]. The elution containing the pooled DNA library was further processed for cluster generation and sequencing using NextSeq 500 High Output kit 75 cycles and Illumina NextSeq 500 sequencing platform, respectively. Sequencing data have been deposited in the Gene Expression Omnibus (GEO) database with accession number GSE114860. The resulting miRNA counts were filtered, and only miRNAs with at least 1 count in every sample were retained. The DESeq2 package (version 1.18.1) in R (version 3.2.2) was used to normalize the raw miRNA counts by applying the median ratio method.
5
Soil DNA Extraction and Purification
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Five grams of soil were used to extract DNA with the DNeasy PowerMax Soil Kit (Qiagen, The Netherlands) according to the manufacturer's protocol. DNA was extracted within 14 days of storage on 4 °C, which is not expected to influence the results (Lauber et al., 2010 ). In addition, to further exclude possible storage effects, DNA of the different replicates was extracted on various days. A negative control (i.e. an empty tube) was included to identify possible contaminations originating from the extraction kit or other downstream applications. All samples were subjected to RNase A treatment (1 U/100 μl) (Qiagen, The Netherlands) and extensively purified as soil samples are known to contain several PCR inhibitors and thus could interfere with 16S rRNA gene sequencing (Schrader et al., 2012 ). Details regarding the purification steps are given in Supporting Information . Finally, the concentration and integrity of the DNA was analysed with the Quantifluor dsDNA sample kit (Promega, the Netherlands) and gel electrophoresis, respectively.
6
Developmental Cardiac Tissue Procurement
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The hearts of C57BL/6J mice at the age of 8 weeks (w8) were purchased from the Jackson Laboratories (Bar Harbor, USA). The hearts of the C57BL/6J embryos (embryonic day 15, 16, 18 and 19 (E15, E16, E18, E19) and murine neonates at the age of 1 day (d1), 7 days (d7) and 2 weeks (w2) were purchased from the Tri-city Experimental Animal Centre - Research and Services Centre - Medical University of Gdańsk (Poland). The tissues were collected on RNA Later (Qiagen, cat. no. 76104) and transported in dry ice. Tissues were disrupted with mortar and pestle in liquid nitrogen and divided into two portions, for RNA and DNA isolation. Genomic DNA was extracted by using DNeasy Blood and Tissue Mini Kit (Qiagen, cat. no. 69504) with RNaseA treatment (Qiagen, cat. no. 19101) for RNA removal. Total RNA was extracted with RNeasy Mini Kit (Qiagen, cat. no. 74104) with on-column DNA digestion with RNase-Free DNase Set Kit (Qiagen, cat. no. 79254).
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