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Authors: Remco Stam

Proper citation: Remco Stam 2020. DNA extraction from plants. protocols.io https:// Copy   

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Authors: Marda Jorgensen
Group: Human BioMolecular Atlas Program (HuBMAP) Method Development Community
Summary: The purpose of this Standard Operating Procedure is to outline procedures for processing and storing spleen received for HuBMAP consortium assay and analysis.

Proper citation: Marda Jorgensen 2020. Case Processing SOP (Spleen). protocols.io https://dx.doi.org/10.17504/protocols.io.bbcuiiww Copy   

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Authors: Laura Sánchez
Group: AEGIS - Madrid iGEM 2019
Summary: This protocol is used to hibridate two complementary DNA chains.

Proper citation: Laura Sánchez 2019. Hybridization of DNA oligos. protocols.io https://dx.doi.org/10.17504/protocols.io.8guhtww Copy   

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Authors: Heather Robeson, Jing Jin, Mohammed S. Orloff
Summary: Used applied biosystems high-capacity RNA-to-cDNA kit and fast SYBR green master mix protocol

Proper citation: Heather Robeson, Jing Jin, Mohammed S. Orloff 2020. RNA to cDNA and RT-PCR protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.bpatmien Copy   

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Authors: Anthony Brusa, Eric Patterson
Summary: This protocol was developed for the detection of Amaranthus palmeriseeds in mixed pools with other Amaranthus sp. seeds. This is a bulk testing protocol; each pool of seeds generates one data point, the presence or absence of A. palmeri in that sample. Testing pools should be assembled based on the relevant information (e.g. location, population, seed lot, seed supplier, etc.). Controls are extremely important for KASP based assays; this protocol requires positive, negative, and no-template controls. We strongly recommend running 8 controls for the rare allele being detected (e.g. A. palmeri) to aid in data analysis.The protocols may be extended to any species for which appropriate species-specific KASP markers have been developed. DNA extraction protocols were performed using the MPBio FastDNA Plant Kit, but other methods may be substituted.

Proper citation: Anthony Brusa, Eric Patterson 2021. Protocol for the detection of rare alleles in bulk seed samples. protocols.io https://dx.doi.org/10.17504/protocols.io.brtam6ie Copy   

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Authors: Andrew Potter
Group: Human Cell Atlas Method Development Community
Summary: This protocol was used to dissociate adult (8-10 wk) mouse lung tissue. The entire procedure is carried out on ice (to reduce artifact gene expression changes) and takes about half an hour. The yield was 16,240 non-RBC/mg tissue with 87% viability. 

Proper citation: Andrew Potter 2018. Adult mouse lung cell dissociation (on ice). protocols.io https://dx.doi.org/10.17504/protocols.io.q6bdzan Copy   

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Authors: Sean Misek
Group: Neubig_Lab

Proper citation: Sean Misek 2018. Routine Cell Culture. protocols.io https://dx.doi.org/10.17504/protocols.io.q2jdycn Copy   

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Authors: Marius Ilié, Paul Hofman
Summary: Optimised VENTANA BenchMark ULTRA protocol for PD-L1 assay using the concentrate 22C3 antibody.For the purpose of protocol development 95% of clinical specimens consisted of surgical resection specimens and the remaining 5% were bronchial biopsies and transbronchial mediastinal lymph node biopsies.

Proper citation: Marius Ilié, Paul Hofman 2017. PD-L1 22C3 LDT on VENTANA BenchMark ULTRA automate. protocols.io https://dx.doi.org/10.17504/protocols.io.ixacfie Copy   

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Authors: Bruce Mcdonald
Group: Zymoseptoria community protocols (STBnet)
Summary: Bruce McDonald 1990 modified from Murray and Thompson

Proper citation: Bruce Mcdonald 2018. Total DNA "Midi" CTAB prep. for Zymoseptoria tritici. protocols.io https://dx.doi.org/10.17504/protocols.io.s2wegfe Copy   

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Authors: Katy Monteith
Group: Vale Lab
Summary: This protocol describes how to make 1 L of PBS to a final concentration of 1x or 10x solution. The solution can be made RNase free with the addition of DEPC (diethyl pyrocarbonate) as in step 5.

Proper citation: Katy Monteith 2018. Phosphate-buffered Saline (PBS). protocols.io https://dx.doi.org/10.17504/protocols.io.p4rdqv6 Copy   

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Authors: Kelsey Knight
Group: BioLegend

Proper citation: Kelsey Knight 2018. Cell Surface Immunofluorescence Staining Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.tihekb6 Copy   

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Authors: Monica Rieth, Andrew Lozano, Jordan Grant
Group: Labyrieth
Summary: This method outlines a general approach for preparing liposomes using probe-tip sonication. The method has been optimized for the preparation of pure DPPC liposomes on a 25-mg scale and may require modifications as the quantity of lipid is altered or upon addition of other lipids and small molecules.

Proper citation: Monica Rieth, Andrew Lozano, Jordan Grant 2020. General preparation of liposomes using probe-tip sonication. protocols.io https://dx.doi.org/10.17504/protocols.io.3tdgni6 Copy   

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Authors: Emmy Li, Rene Sit
Group: Chan Zuckerberg Biohub

Proper citation: Emmy Li, Rene Sit 2019. Nuclei Isolation Prep and Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.6m8hc9w Copy   

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Authors: Stephen Floor
Group: Stephen Floor Lab
Summary: Standard protocol to transform bacteria with a plasmid using chemically competent E. coli and antibiotic resistance.

Proper citation: Stephen Floor 2018. Bacterial transformation. protocols.io https://dx.doi.org/10.17504/protocols.io.mnwc5fe Copy   

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Authors: David Milner
Group: Richards Lab
Summary: Testing for metal toxicity as a result of addition of a putative phosphate transporter to a S. cerevisiae Δpho84 strain

Proper citation: David Milner 2018. S. cerevisiae Δpho84 complementation: metal toxicity. protocols.io https://dx.doi.org/10.17504/protocols.io.re4d3gw Copy   

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Authors: Aaron Bivins, Warish Ahmed, Devin North, Kyle Bibby
Group: Coronavirus Method Development Community, 2019-nCoV Wastewater Epidemiology
Summary: The following protocol describes the GERM Lab workflow for processing wastewater for SARS-CoV-2 via pH adjustment, MCE filtration, extraction, and assay by droplet digital PCR (ddPCR). It is synonymous to method A as described in Ahmed et al. 2020 (the referenced manuscript) with modifications to the consumables and PCR format. It is intended for use in a BSL2+ lab with extra precautions for processing environmental specimens for SARS-CoV-2 as described in the CDC Interim Laboratory Biosafety Guidelines.

Proper citation: Aaron Bivins, Warish Ahmed, Devin North, Kyle Bibby 2020. Wastewater Concentration by Adsorption and Direct Extraction for SARS-CoV-2 RNA Detection and Quantification using RT-ddPCR. protocols.io https://dx.doi.org/10.17504/protocols.io.bhiuj4ew Copy   

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Authors: Jialing Lin, Zhenjiang Yao
Summary: A cross-sectional study was conducted in eight elementary schools in Guangzhou, China. Nasal swabs from students and environmental swabs from school environments were collected. Univariate and multivariate logistic regression analyses under a multistage stratified cluster cross-sectional survey design were performed to access the prevalence relationship and influencing factors, respectively. Phenotypic and molecular characterizations of MSSA isolates were conducted using the Kirby-Bauer disk diffusion method and polymerase chain reaction assays, respectively.

Proper citation: Jialing Lin, Zhenjiang Yao 2019. School environmental contamination of methicillin-sensitive Staphylococcus aureus as an independent risk factor for nasal colonization in schoolchildren: an observational, cross-sectional study protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.wqvfdw6 Copy   

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Authors: Dante Heredia, Thomas Gould
Summary: Protocol for optogenetically stimulating neuronal subtypes in murine colonic myenteric plexus.

Proper citation: Dante Heredia, Thomas Gould 2020. Optogenetically stimulating enteric neurons in the murine large intestine.. protocols.io https://dx.doi.org/10.17504/protocols.io.bgr9jv96 Copy   

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Authors: Amy Lyden, Emily Crawford, Jenai Quan, Saharai Caldera, David Dynerman
Group: Chan Zuckerberg Biohub
Summary: This protocol is for performing Depletion of Abundant Sequences by Hybridization (DASH) after preparing sequencing libraries and pooling together.DASH is most useful for RNA-seq of human metagenomic samples, where abundant species such as human mitochondrial ribosomal RNAs (rRNAs) occupy a majority of the sequencing space available, leaving a minor fraction for regions of interest. DASH treatment is performed after ligation of adapters and unique barcoding of the RNA-seq library. It employs CRISPR-Cas9 complexed to a set of guide RNAs (gRNAs) targeted to the abundant regions to be depleted in a given library. These abundant regions in the library are then cleaved, leaving only the fragments with intact adapters on both ends to be further amplified and sequenced.

Proper citation: Amy Lyden, Emily Crawford, Jenai Quan, Saharai Caldera, David Dynerman 2019. DASH Protocol v2.5. protocols.io https://dx.doi.org/10.17504/protocols.io.y9gfz3w Copy   

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Authors: Leandro Xavier Neves, R. Alan Wilson, William de Castro Borges
Summary: Schistosomes are intravenous parasites with ability to survive in the mammalian host for decades, using its blood as a source of nutrients. The feeding process is multistep and takes place along the worm’s alimentary tract, which comprises an (i) oral cavity opening to a short (ii) esophagus that is connected to the (iii) the gut caecum. The ultrastructureal morphology of Schistosoma mansoni and S. japonicum has revealed the existence of two secretory cell masses surrounding the esophagus tube, referred to as the anterior and posterior esophageal glands (antESO and postESO, respectively). We recently established that the esophageal glands have a pivotal role in the first steps of blood processing. For instance, erythrocytes and leucocytes are quickly processed along the esophagus before they are propelled to the lower parts of the intestines for further digestion nutrient uptake. We propose that incorrect functioning of alimentary tract is associated with worms death by starvation. This was first observed in the self-cure response of Rhesus macaque (Macaca mulatta), one of few known vertebrate hosts capable of combating the disease through worm elimination once the infection is stablished. Classical immunoproteomics (2D-PAGE and Western blotting) has revealed potential targets in both exposed tegument and secreted gut proteins. Recently, a more detailed investigation using S. japonicum in the Rhesus model shed light on the possible operating mechanisms that prevent parasite feeding on blood. In addition, ultrastructural studies and immunocytochemistry on surviving worms indicated the esophageal lumen and the gland secretions as the primary targets of a potent and protective humoral immune response that ultimately disrupts the esophageal functions. Therefore, the molecular characterisation of the esophageal gland constituents is imperative if one intends to emulate the Rhesus self-cure response for therapeutic purposes. However, this is not a trivial task as challenges are multiple. Perhaps, the most important caveat is that both anterior and posterior parts of the oesophageal gland represent a minor fraction of the whole parasite body (or even of its head), meaning that dominant constituents, such as those derived from muscle tissues, suppress the identification of a unique set of gland products. Indeed, our recent investigation on the soluble protein composition of a S. mansoni preparation failed to detect the presence of gland products attesting for their low abundance in the whole worm. Although optimized protocols for chemical/enzymatic dissection are reported for isolation of testes and ovary of adult worms, no method has proved feasible for gastrodermal epithelium and the esophageal gland cells. We tackled these challenges by developing a dissection technique on worms preserved in RNAlater solution aided by an essential set of scissors and tweezers that delivers adequate precision during the procedure. The method herein described is compatible with downstream proteomic analyses using mass spectrometry and has permitted a large-scale characterisation of proteins expressed in the S. mansoni esophagus and gastrodermis. This methodology can be applied in the molecular characterisation of other schistosome organs and tissues that present a well-defined anatomic location.

Proper citation: Leandro Xavier Neves, R. Alan Wilson, William de Castro Borges 2019. Manual dissection of the Schistosoma mansoni esophagus and back end for proteomic analysis. protocols.io https://dx.doi.org/10.17504/protocols.io.tq2emye Copy   

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