Protocols   Select Another Resource Report Type

Authors: Daniel B Nothaft
Group: Chemistry Method Development Community
Summary: CO2 conversion by H3PO4, extracted CO2 introduced into continous flow IRMS for 13C/12C analysisThis method follows the general principles described in Assayag et al. (2006), which emphasizes the importance of maintaining the same water : H3PO4 volume ratio for standard solutions and samples (ratio of 10 in this case) in order to obviate issues regarding isotopic fractionation between CO2 (g) and CO2 (aq).References Assayag, N., Rivé, K., Ader, M., Jézéquel, D., and Agrinier, P. (2006). Improved method for isotopic and quantitative analysis of dissolved inorganic carbon in natural water samples. Rapid Communications in Mass Spectrometry 20, 2243–2251

Proper citation: Daniel B Nothaft 2019. Dissolved inorganic carbon concentration and 13C/12C. protocols.io https://dx.doi.org/10.17504/protocols.io.zduf26w Copy   

•   Source:

Authors: Chin Yee Tan
Summary: A brief protocol for electrotransformation of Clostridium species

Proper citation: Chin Yee Tan 2019. Electrotransformation of Clostridium species. protocols.io https://dx.doi.org/10.17504/protocols.io.zvsf66e Copy   

•   Source:

Authors: Tobias von der Haar
Summary: This protocol is our lab's version of the standard lithium acetate-based transformation protocol originally developed by the Gietz lab (see eg Gietz and Schiestl 2007).

Proper citation: Tobias von der Haar 2017. Rapid Yeast Transformation. protocols.io https://dx.doi.org/10.17504/protocols.io.g8qbzvw Copy   

•   Source:

Authors: James Thornton
Group: Hurwitz Lab, MetaFunc Course
Summary: This protocol provides a procedure to generate taxonomic data from assembled contigs using centrifuge. 

Proper citation: James Thornton 2016. MG_HW7: Taxonomic Classification Using Centrifuge. protocols.io https://dx.doi.org/10.17504/protocols.io.f8qbrvw Copy   

•   Source:

Authors: Jocelyn Y. Kishi, Sylvain W. Lapan, Brian J Beliveau, Emma R. West, Allen Zhu, Hiroshi M. Sasaki, Sinem Saka, Yu Wang, Constance L Cepko, Peng Yin
Group: Human Cell Atlas Method Development Community

Proper citation: Jocelyn Y. Kishi, Sylvain W. Lapan, Brian J Beliveau, Emma R. West, Allen Zhu, Hiroshi M. Sasaki, Sinem Saka, Yu Wang, Constance L Cepko, Peng Yin 2020. 2: User-friendly protocol: Oligo ordering and preparation (SABER-FISH). protocols.io https:// Copy   

•   Source:

Authors: Chunman Zuo, Yuhong Tang, Hao Fu, Yiming Liu, Xunzhong Zhang, Bingyu Zhao, Ying Xu

Proper citation: Chunman Zuo, Yuhong Tang, Hao Fu, Yiming Liu, Xunzhong Zhang, Bingyu Zhao, Ying Xu 2018. Elucidation and Analyses of the Regulatory Networks of Upland and Lowland Ecotypes of Switchgrass in Response to Drought and Salt Stresses. protocols.io https://dx.doi.org/10.17504/protocols.io.saxeafn Copy   

•   Source:

Authors: Seyfullah Yilmaz, C. Yunus Sahan, Serhat Sevli
Summary: Nehir Biyoteknoloji Ltd. www.nehirbt.com This protocol describes the extreme high height microfluidic channel fabrication. In this case the height of the channels is 500 µm and this required some modifications on the previous protocol.

Proper citation: Seyfullah Yilmaz, C. Yunus Sahan, Serhat Sevli 2017. Microfluidics Lithography 6: High Microchannel Thickness (500 µm). protocols.io https://dx.doi.org/10.17504/protocols.io.g9tbz6n Copy   

•   Source:

Authors: Benjamin Izar, Parin Shah, Mei-Ju Su, Isaac Wakiro, Sara Napolitano, Jingyi Wu, Sébastien Vigneau, Asaf Rotem, Orit Rozenblatt-Rosen, Bruce Johnson, Aviv Regev
Group: NCIHTAN
Summary: This protocol is used to process human ovarian cancer ascites to a single-cell suspension compatible with droplet-based single-cell RNA-Seq technology.A majority of ascites samples have a high proportion (>90%) of CD45-expressing (CD45+) cells, thus greatly reducing the ability to capture non-immune and malignant cells. To address this issue, we have included a CD45+ cell depletion step to reduce the number of CD45+ cells. In such samples, where the proportion of immune cells is very high, a large proportion of both CD45+ and CD45- cells can be recovered post-depletion, allowing simultaneous assessment of the immune and non-immune microenvironment. In cases with a lower initial proportion of CD45+ cells, however, non-immune cells tend to be dominant following CD45+ cell depletion.Of note, many ascites samples contain multicellular aggregates, or spheroids, which pose challenges during sample processing and dissociation. Larger aggregates are removed through an initial 70 μm straining (Step 14) whereas smaller residual aggregates, when present, can be removed through a final 30 μm straining (optional Step 20). Yet, we have encountered instances where small aggregates cannot be efficiently removed through straining. This protocol is not optimized for those cases. In addition, we would like to note that ascites can be quite variable and further optimization of this protocol may be needed to handle cases with atypical characteristics. For the Human Tumor Atlas Pilot Project (HTAPP), this protocol was successfully applied to 4 ovarian cancer ascites samples. Description of this protocol and guidance for testing and selecting methods for processing other tumor and sample types can be found in Slyper et al.

Proper citation: Benjamin Izar, Parin Shah, Mei-Ju Su, Isaac Wakiro, Sara Napolitano, Jingyi Wu, Sébastien Vigneau, Asaf Rotem, Orit Rozenblatt-Rosen, Bruce Johnson, Aviv Regev 2020. HTAPP_Processing human ovarian cancer ascites to a single-cell suspension for single-cell RNA-seq. protocols.io https://dx.doi.org/10.17504/protocols.io.bhbkj2kw Copy   

•   Source:

Authors: Solize Vosloo, Maria Sevillano, Ameet Pinto
Group: Pinto Lab
Summary: DNA-extractions from drinking water samples are essential for a range of subsequent microbial community quantitation and characterization methods, i.e., quantitative polymerase chain reaction (qPCR) assays targeting specific genes and the characterization of compositional and functional profiles using high throughout sequencing technologies (e.g. amplicon sequencing and shotgun metagenomic sequencing). Despite advances in the specificity and sensitivity of molecular techniques, efficient recovery of DNA from drinking water samples, particularly those with low cell counts, remains challenging. Drinking water samples, in which microbial concentrations range between 103and 105 cells.ml-1, generally requires the collection of large volume of sample and subsequent processing by filtration to concentrate microbial cells. Here we document a modified version of the DNeasy PowerWater Kit® protocol that utilizes enzymatic, chemical, and mechanical lysis strategies to enhance recovery of DNA from drinking water samples. The DNA quantities recovered using this protocol are typically at least two to three-fold higher when compared to the routine DNeasy PowerWater Kit® protocol. In our hands, this protocol consistently provides sufficient DNA of high quality from as little as 1.5 liters of filtered drinking water with cell counts in the range of 103-104 cells.ml-1, while maintaining the 16S rRNA qPCR counts at least 100-1000 times higher compared to DNA extracts from negative controls (i.e., blank unused filters, filters with autoclaved deionized water filtered, and reagent blanks) processed identically as the drinking water samples. .justify:after { content: ""; display:inline-block; width: 100%; }

Proper citation: Solize Vosloo, Maria Sevillano, Ameet Pinto 2019. Modified DNeasy PowerWater Kit® protocol for DNA extractions from drinking water samples. protocols.io https://dx.doi.org/10.17504/protocols.io.66khhcw Copy   

•   Source:

Authors: Eric Pederson
Summary: This is a detailed protocol concerning the DNA extraction from the genome paper titled "Genome Sequencing of Pleurozium schreberi: The Assembled and Annotated Draft Genome of a Pleurocarpous Feather Moss" (https://doi.org/10.1534/g3.119.400279).

Proper citation: Eric Pederson 2020. Pleurozium schreberi (moss) DNA extraction. protocols.io https://dx.doi.org/10.17504/protocols.io.7ekhjcw Copy   

•   Source:

Authors: Yu Xi, Huijing Liu, Yuqiang Zhao, Ji Li, Wenchao Li, Guorong Liu, Jiayong Lin, Wanghong Liu, Jinlong Zhang, Minggang Lei, Debin Ni
Summary: Tongcheng pigs (TC) and Yorkshire (YK) are two pig breeds with distinguished morphologies in muscle. A comprehensive study of porcine microRNAome (miRNAome) in longissimus muscle during 5 developmental stages (40, 55, 63, 70 and 90 dpc (days post coitum)) using Solexa sequencing technology, was carried out in order to compare miRNA expression involved in the differentiated regulation of skeletal muscle development between the TC and YK breeds. Known miRNAs and novel miRNAs were identified by alignment and annotation. MiRNA expression profiles were normalized by transcript per million (TPM). Principal component analysis (PCA) and hierarchical cluster analysis (HCA) were used to cluster the samples based on the expression values of miRNAs. The short time-series expression miner (STEM) was conducted in order to further explore the temporal expression characteristics of miRNAs in the TC and YK breeds. Then the analysis of differentially expressed (DE) miRNAs was performed. Functional annotation of the predicted miRNA targets was performed based on Gene Ontology slim database (GO-Slim) and Kyoto Encyclopedia of Genes and Genomes database (KEGG). Ssc-miR-499-5p is the only multipoint (no less than 4 time points)-DE miRNA with considerable expression level during the time periods investigated. Combining the target prediction and dual luciferase reporter assay, we validated DSTN was the target gene of ssc-miR-499-5p. The results are expected to facilitate the understanding of the differences of miRNAs in myogenesis between the TC and YK breed and provide implications for the improvement of meat quality in animal production.

Proper citation: Yu Xi, Huijing Liu, Yuqiang Zhao, Ji Li, Wenchao Li, Guorong Liu, Jiayong Lin, Wanghong Liu, Jinlong Zhang, Minggang Lei, Debin Ni 2018. Comparative analyses of miRNAomes between Tongcheng and Yorkshire Pigs. protocols.io https://dx.doi.org/10.17504/protocols.io.mpkc5kw Copy   

•   Source:

Authors: Adriana Alberti, Julie Poulain, Stefan Engelen, Karine Labadie, Sarah Romac, Isabel Ferrera, Guillaume Albini, Jean-Marc Aury, Caroline Belser, Alexis Bertrand, Corinne Cruaud, Corinne Da Silva, Carole Dossat, Frédéric Gavory, Shahinaz Gas, Julie Guy, Maud Haquelle, E'krame Jacoby, Olivier Jaillon, Arnaud Lemainque, Eric Pelletier, Gaëlle Samson, Marc Wessner, Genoscope Technical Team, Silvia G. Acinas, Marta Royo-Llonch, Francisco M. Cornejo-Castillo, Ramiro Logares, Beatriz Fernández-Gómez, Chris Bowler, Guy Cochrane, Clara Amid, Petra Ten Hoopen, Colomban De Vargas, Nigel Grimsley, Elodie Desgranges, Stefanie Kandels-Lewis, Hiroyuki Ogata, Nicole Poulton, Michael E. Sieracki, Ramunas Stepanauskas, Matthew B. Sullivan, Jennifer R. Brum, Melissa B. Duhaime, Bonnie T. Poulos, Bonnie L. Hurwitz, Stéphane Pesant, Eric Karsenti, Patrick Wincker
Group: Tara Oceans
Summary: This protocol describes the nucleic acids preparations for the Tara Oceans expedition and is part of Viral to metazoan marine plankton nucleotide sequences from the Tara Oceans expedition. Figure 1: Overview of -omics analysis strategy applied on Tara Oceans samples.

Proper citation: Adriana Alberti, Julie Poulain, Stefan Engelen, Karine Labadie, Sarah Romac, Isabel Ferrera, Guillaume Albini, Jean-Marc Aury, Caroline Belser, Alexis Bertrand, Corinne Cruaud, Corinne Da Silva, Carole Dossat, Frédéric Gavory, Shahinaz Gas, Julie Guy, Maud Haquelle, E'krame Jacoby, Olivier Jaillon, Arnaud Lemainque, Eric Pelletier, Gaëlle Samson, Marc Wessner, Genoscope Technical Team, Silvia G. Acinas, Marta Royo-Llonch, Francisco M. Cornejo-Castillo, Ramiro Logares, Beatriz Fernández-Gómez, Chris Bowler, Guy Cochrane, Clara Amid, Petra Ten Hoopen, Colomban De Vargas, Nigel Grimsley, Elodie Desgranges, Stefanie Kandels-Lewis, Hiroyuki Ogata, Nicole Poulton, Michael E. Sieracki, Ramunas Stepanauskas, Matthew B. Sullivan, Jennifer R. Brum, Melissa B. Duhaime, Bonnie T. Poulos, Bonnie L. Hurwitz, Stéphane Pesant, Eric Karsenti, Patrick Wincker 2020. Nucleic acids preparations. protocols.io https://dx.doi.org/10.17504/protocols.io.qwgdxbw Copy   

•   Source:

Authors: Jacob Kirkland
Summary: The best way to determine TOP2 specific accessibility to chromatin is via etoposide treatment, which leads to a covalent cross-link between TOP2 proteins and DNA, that can only occur where TOP2 proteins have access to DNA. TOP2 cross-linking to DNA has previously been shown to require DNA accessibility and chromatin regulators that alter DNA accessibility (Dykhuizen and Hargreaves et al. Nature 2013, Miller et al. NSMB 2017).

Proper citation: Jacob Kirkland 2019. Top2 Chromatin Accessibility by Etoposide Cross-linking. protocols.io https://dx.doi.org/10.17504/protocols.io.2rngd5e Copy   

•   Source:

Authors: Adriana Alberti
Group: VERVE Net, Sullivan Lab
Summary: Protocol for preparation of short single and paired-end libraries from genomic dsDNA starting from low DNA quantity (up to 10 ng) for Illumina sequenincg. Developed by Adriana Alberti at Genoscope.

Proper citation: Adriana Alberti 2016. Low input Library Preparation for Illumina Sequencing. protocols.io https://dx.doi.org/10.17504/protocols.io.ddq25v Copy   

•   Source:

Authors: Julie Huber, Caroline Fortunato
Group: Center for Dark Energy Biosphere Investigations, Huber Lab
Summary: This is a DNA extraction protocol adapted from Byron Crump's lab, which was developed from both Zhou et al. 1996 and Crump et al. 2003. It is intended to be used with 47 mm 0.22 um PES filters or a 0.22 uM Sterivex filter, both preserved with RNALater after sample collection.

Proper citation: Julie Huber, Caroline Fortunato 2017. DNA Extraction from Filtered Vent/Crustal Fluids or Seawater. protocols.io https://dx.doi.org/10.17504/protocols.io.iuwcexe Copy   

•   Source:

Authors: Sonia M. Hall

Proper citation: Sonia M. Hall 2015. Optimized Concentrations for Developmental Studies Hybridoma Bank and Custom Antibodies for use with Drosophila Embryos. protocols.io https://dx.doi.org/10.17504/protocols.io.daq2dv Copy   

•   Source:

Authors: Mickey Shah, Gary Niehaus
Group: Coronavirus Method Development Community, XPRIZE Rapid Covid Testing
Summary: The Crystal Diagnostics AutoXpress provides rapid detection of COVID 19 at 10 - 100 CFU per ul of clinical sample (nasal swab, sputum, or saliva). The patented Liquid Crystal technology is combined with antibody-coated paramagnetic microspheres to selectively capture and detect COVID 19.

Proper citation: Mickey Shah, Gary Niehaus 2020. CDx autoXpress detection of COVID 19. protocols.io https://dx.doi.org/10.17504/protocols.io.bki4kugw Copy   

•   Source:

Authors: Allan Gonzalez, Jiri Hulcr
Group: Protocols Bark Beetle Mycobiome
Summary: This protocol describes how to safely transport bark and ambrosia beetles. This protocol is part of the Bark Beetle Mycobiome (BBM) Research Coordination Network. For more information on the BBM international network: Hulcr J, Barnes I, De Beer ZW, Duong TA, Gazis R, Johnson AJ, Jusino MA, Kasson MT, Li Y, Lynch S, Mayers C, Musvuugwa T, Roets F, Seltmann KC, Six D, Vanderpool D, & Villari C. 2020. Bark beetle mycobiome: collaboratively defined research priorities on a widespread insect-fungus symbiosis. Symbiosis 81: 101–113 https://doi.org/10.1007/s13199-020-00686-9.

Proper citation: Allan Gonzalez, Jiri Hulcr 2020. Keeping beetles alive in transport with wood-flour media. protocols.io https://dx.doi.org/10.17504/protocols.io.bny2mfye Copy   

•   Source:

Authors: Kelsey Miller, Sam Li
Group: BioLegend
Summary: BioLegend MojoSort™ nanobeads work in commonly used separation columns, based on our internal research as well as validation by external testing by academic labs. This simple protocol consists of following the MojoSort™ protocol to label the cells with pre-diluted MojoSort™ reagents and using the columns as indicated by the manufacturer.Note: Due to the properties of our beads, it may be possible to use far fewer beads and less antibody cocktail that with other commercial suppliers. We recommend a titration to find the best dilution factor. However, as a general rule, dilutions ranging from 1:2 to 1:10 for the antibody cocktail can be used. Dilutions ranging from 1:5 to 1:20 for the Streptavidin Nanobeads can be used. Please contact BioLegend Technical Service ([email protected]) if further assistance is needed.

Proper citation: Kelsey Miller, Sam Li 2019. MojoSort™ Streptavidin Nanobeads Column Protocol - Negative Selection. protocols.io https://dx.doi.org/10.17504/protocols.io.693hh8n Copy   

•   Source:

Authors: Tobias Wandt

Proper citation: Tobias Wandt 2020. DNA-Extraction-ClostridiaProject. protocols.io https:// Copy   

•   Source: