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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. 4: User-friendly protocol: Cost-efficient Primer Exchange Reaction (PER) concatemerization (SABER-FISH). protocols.io https:// Copy   

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Authors: Addgene The Nonprofit Plasmid Repository
Summary: This protocol describes plasmid cloning by Polymerase Chain Reaction (PCR). To see the full abstract and additional resources, please visit the Addgene protocol page.

Proper citation: Addgene The Nonprofit Plasmid Repository 2019. Plasmid Cloning by PCR. protocols.io https://dx.doi.org/10.17504/protocols.io.43rgym6 Copy   

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Authors: Jakub Nedbal
Summary: The LED controller electronics regulates the LED current, adjusting the irradiance of the sample according the experimental needs. It also powers the fans cooling down the LEDs.This document is part of the Illuminated Orbital Shaker for Microalgae Culture project:​Procuring Parts for Algal Shaker​​Assembling LED Controller Electronics (this document)​3D Printing Case for LED Controller​​Assembling Cooled LED Illuminator​​Cutting and Drilling Clear Acrylic Sheet​​Assembling Algal Shaker

Proper citation: Jakub Nedbal 2020. Assembling LED Controller Electronics. protocols.io https://dx.doi.org/10.17504/protocols.io.bcfkitkw Copy   

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Authors: William H. Wilson and Declan Schroeder
Group: VERVE Net
Summary: Generation of DNA fragments by DNase digestion involves digesting DNA for a range of times, then picking the time that gives optimal-sized DNA fragments (typically 1000–4000 bp).

Proper citation: William H. Wilson and Declan Schroeder 2016. Generation of DNA fragments by DNase digestion. protocols.io https://dx.doi.org/10.17504/protocols.io.dy47yv Copy   

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Authors: Alexandra Netter-Glangeaud
Group: StressMarq

Proper citation: Alexandra Netter-Glangeaud 2019. Tau Thioflavin T Assay. protocols.io https://dx.doi.org/10.17504/protocols.io.wxgffjw Copy   

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Authors: Izabela Rezende, Lívia Sacchetto
Group: MRCA
Summary: For investigate the presence of YFV genome by quantitative PCR assays in the hydrolysis probe detection format. The target is the 5’-noncoding region (5’-NC) of the YFV genome (Domingo et al., 2012).

Proper citation: Izabela Rezende, Lívia Sacchetto 2018. Real-time Reverse Transcription Polymerase Chain Reaction (RT- qPCR). protocols.io https://dx.doi.org/10.17504/protocols.io.pw8dphw Copy   

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Authors: Vasilios Zachariadis, Huaitao Cheng, Nathanael Andrews, Martin Enge
Group: Enge lab
Summary: Understanding how genetic variation alters gene expression - how genotype affects phenotype - is a central challenge in biology. To address this question in complex cell mixtures, we developed Direct Nuclear Tagmentation and RNA-sequencing (DNTR-seq), which enables whole genome and mRNA sequencing jointly in single cells.

Proper citation: Vasilios Zachariadis, Huaitao Cheng, Nathanael Andrews, Martin Enge 2020. Direct nuclear tagmentation and RNA-sequencing (DNTR-seq). protocols.io https://dx.doi.org/10.17504/protocols.io.bdthi6j6 Copy   

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Authors: Tanya Puccio, Todd Kitten
Summary: Streptococcus sanguinis is a lactic acid-forming bacterium that can be cultured in both aerobic and anaerobic conditions. It is a primary colonizer of the oral cavity but can also cause a heart disease called infective endocarditis. Our main objective for this protocol was to grow large, controlled cultures before and after manganese depletion for various analyses. In order to obtain large scale, reproducible growth of S. sanguinis, a biostat was used to maintain controlled conditions. After optimization, it was determined that traditional chemostat conditions (Burne and Chen, 1998) were not appropriate for these experiments. Thus, we decided on this modified protocol. Using this method, we were able to identify a concentration of the non-specific metal chelator, EDTA, that would lead to a decreased growth rate in a manganese transporter mutant but not in the wild-type strain.

Proper citation: Tanya Puccio, Todd Kitten 2020. Fermentor Growth of Streptococcus sanguinis. protocols.io https://dx.doi.org/10.17504/protocols.io.bkayksfw Copy   

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Authors: Lukas Beule

Proper citation: Lukas Beule 2020. Protocol for DNA extraction from clay-rich subsoils. protocols.io https://dx.doi.org/10.17504/protocols.io.bhmjj44n Copy   

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Authors: Addgene The Nonprofit Plasmid Repository
Summary: This protocol describes ddPCR titration of Lentivirus vectors. To see the full abstract and additional resources, visit https://www.addgene.org/protocols/lentivirus-ddpcr-titration/.This protocol was modified from the publication Wang Y, Bergelson S, Feschenko M, 2018. Sample Data: When analyzing data there should be a clear distinction between negative droplets (black) and positive droplets (blue/green).The concentration of RRE positive droplets in the untransduced control should be close to zero (A01).In this protocol, the lentiviral particles are serially diluted and used to transduce HEK293T cells. Genomic DNA is extracted from the target cells and assayed for integrated copies of RRE. Since the samples that are assayed are diluted 2-fold serially, the concentration of RRE positive droplets should decrease by a factor of 2 across the dilutions. RPP30 copies should be relatively constant across samples.In the RRE example below, 2-fold serial dilutions of a sample were loaded in wells B01-H01.As shown in the image and table below, the concentration of RRE positive droplets increases by a factor of ~2 as you progress from the higher dilutions to the lower dilutions (blue).The concentration of RPP30 positive droplets stays relatively even across samples (green).To increase the accuracy of the titer, calculate and average of several dilutions.

Proper citation: Addgene The Nonprofit Plasmid Repository 2020. ddPCR Titration of Lentivirus Vectors. protocols.io https://dx.doi.org/10.17504/protocols.io.be7ijhke Copy   

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Authors: Daniel Vaulot
Group: Ecology of Marine Plankton (ECOMAP) team - Roscoff
Summary: Protocol to fix marine samples for flow cytometry sorting of phytoplankton. Fix at least 2 samples per depth sampled and at least 6 to 10 depths per vertical profile.Back to the laboratory, pico and nano-plankton populations can be sorted and used for clone library construction or metabarcoding with Next Generation Sequencing (e.g. Illumina).  This was successfully tested on samples from a cruise off Brazil (unpublished data)ReferenceRibeiro C., Lopes A., Marie D., Vaulot D. Unpublished data

Proper citation: Daniel Vaulot 2015. Fixation of marine samples for flow cytometry sorting. protocols.io https://dx.doi.org/10.17504/protocols.io.d2x8fm Copy   

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Authors: Geoffrey W. Meissner, Jonathan B. Grimm, Rebecca M. Johnston, Ben Sutcliffe, Julian Ng, Gregory S.X.E. Jefferis, Sebastian Cachero, Luke D. Lavis, Oz Malkesman
Summary: The use of genetically encoded ‘self-labeling tags’ with chemical fluorophore ligands enables rapid labeling of specific cells in neural tissue. To improve the chemical tagging of neurons, we synthesized and evaluated new fluorophore ligands based on Cy, Janelia Fluor, Alexa Fluor, and ATTO dyes and tested these with recently improved Drosophila melanogaster transgenes. We found that tissue clearing and mounting in DPX substantially improves signal quality when combined with specific non-cyanine fluorophores. We compared and combined this labeling technique with standard immunohistochemistry in the Drosophila brain.

Proper citation: Geoffrey W. Meissner, Jonathan B. Grimm, Rebecca M. Johnston, Ben Sutcliffe, Julian Ng, Gregory S.X.E. Jefferis, Sebastian Cachero, Luke D. Lavis, Oz Malkesman 2018. Polarity IHC of Drosophila CNS. protocols.io https://dx.doi.org/10.17504/protocols.io.qf6dtre Copy   

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Authors: BioLegend, Inc., Kelsey Knight
Group: BioLegend

Proper citation: BioLegend, Inc., Kelsey Knight 2018. Immunocytochemistry Staining Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.tdmei46 Copy   

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Authors: Benjamin Schwessinger and Megan McDonald
Group: Solomon Lab - Australian National University, High molecular weight DNA extraction from all kingdoms, Zymoseptoria community protocols (STBnet)
Summary: Extraction of high quality DNA for long read sequencing e.g. PacBio and MinIonOptimized for DNA extraction from Bipolaris sorokiniana. Also tested on Parastaognospora nodorum, Zymoseptoria tritici, wheat stripe rust, barley stripe rust and Pyrenophora tritici-repentisBuffers are best when fresh and not older than 3-6 months. Buffered Phenol:Chloroform:Isoamylalcohol (25:24:1) should not be older than 3 months.Critical steps to obtain high quality DNA:Do NOT heat samples during DNA extractions! Perform all steps at RT or 4oC as indicated.Do NOT incubate samples with KAc for prolonged time periodsPerform two steps of buffered Phenol:Chloroform:Isoamylalcohol purification to reduce co-purifying metabolites.DNA fragments were well above the 40kb mark based on Pippin Pulse Gels. The sequencing center performed a second AMPure purification step before library construction. 

Proper citation: Benjamin Schwessinger and Megan McDonald 2017. High quality DNA from Fungi for long read sequencing e.g. PacBio, Nanopore MinION. protocols.io https://dx.doi.org/10.17504/protocols.io.k6qczdw Copy   

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Authors: Allen Institute for Brain Science
Group: BICCN, Allen Institute for Brain Science
Summary: This protocol is used for in vitro transcription (IVT) reaction generating DIG-labeled anti-sense (AS) RNA from polymerase chain reaction (PCR) product for in situ hybridization (ISH), as well as fluorescent quantification of products using Ribogreen immediately following. This protocol is written for 96 reactions but can also be used for single reactions or in multiples of 96 by adjusting the amount of materials and equipment appropriately.Note: Research reported in this publication was supported by the National Institute Of Mental Health of the National Institutes of Health under Award Number U19MH114830. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Proper citation: Allen Institute for Brain Science 2020. Digoxigenin Labeled In Vitro Transcription and Ribogreen Quantification. protocols.io https://dx.doi.org/10.17504/protocols.io.bg4vjyw6 Copy   

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Authors: Dr. Steven Wilhelm
Group: The Aquatic Microbial Ecology Research Group - AMERG (The Buchan, Zinser and Wilhelm labs)
Summary: Please contact Dr. Steven Wilhelm ([email protected]) for more information regarding this protocol.Modified from Pardee, A. B., F. Jacob, and J. Monod. 1959. The genetic control and cytoplasmic expression of "inducibility" in the synthesis of ß-galactosidase in E. coli. J. Mol. Biol. 1:165-178

Proper citation: Dr. Steven Wilhelm 2017. M9 Salts. protocols.io https://dx.doi.org/10.17504/protocols.io.ibwcape Copy   

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Authors: Wen Aw
Group: Cage Studies

Proper citation: Wen Aw 2018. Triglyceride Quantification. protocols.io https://dx.doi.org/10.17504/protocols.io.rv9d696 Copy   

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Authors: Chee Lim
Group: Mouse Metabolic Phenotyping Centers
Summary: Summary:The tail-cuff blood pressure measurement is a non-invasive test and the principle is the same as that of the inflatable blood pressure cuff used in the standard clinic visit. The Visitech system uses an LED light source to detect the pulse signal wave. Since the measurement is sensitive to motion artifact, the procedure requires training sessions to acclimate the mouse to the process.

Proper citation: Chee Lim 2019. Vandy - Tail-Cuff Blood Pressure. protocols.io https://dx.doi.org/10.17504/protocols.io.yymfxu6 Copy   

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Authors: Irina Agarkova
Group: VERVE Net, The Chlorovirus Group

Proper citation: Irina Agarkova 2016. Pulse Field Gel Electrophoresis (PFGE) Protocol for separation of Chlorella chromosomal DNA and Chlorella virus DNA. protocols.io https://dx.doi.org/10.17504/protocols.io.essbeee Copy   

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Authors: Ning Ding
Summary: Forty SD male rats were divided into five groups randomly (n = 8 per group): the normal control (N) group, the normal saline group (NS), the light osteoarthritis group (A), the mild osteoarthritis group (B) and the heavy osteoarthritis group (C). On day 0, the rats in the A, B and C group were injected with 0.3, 1, 3 mg MIA respectively, and rats in the NS group were injected with 50 μl of saline solution.The tissues at the acupoints Yanglingquan (GB34), Heding (EX-LE2) and Weizhong (BL40) were dissected. The dimensions of each tissue fragment were 1.5 × 1.5 × 1.5 mm. The tissues were fixed in 10% neutral formalin, dehydrated, embedded in paraffin and sectioned with a 4-μm slicer. Subsequently, the sections were dewaxed, dehydrated and stained in 0.5% toluidine blue for 30 min and then washed with tap-water.the knee joints of 6 rats in each group were fixed in 10% neutral formalin after samples of GB34, EX-LE2 and BL40 were dissected. Then, the knee joints were placed in 5% formic acid for demineralization, dehydrated, embedded in paraffin and sectioned with a 5-μm slicer. Subsequently, the sections were dewaxed, dehydrated, and stained in 1% fast green for 1.5 min and then differentiated by acetic acid. Next, the sections were stained by 0.5% safranine-O for 1.5 min, differentiated by ethanol, and washed with tap-water. The tissues at GB34, EX-LE2, and BL40 were dissected, fixed in 4% paraformaldehyde for 2.5 h, and dehydrated by 25% sucrose for 2 days. A frozen 10-μm section was sliced by a freezing microtome. For staining, the sections were washed in 0.1 M PB (pH=7.4) and blocked in 0.1 M PB (pH=7.4) containing 3% normal donkey serum and 0.5% Triton X-100 for 30 min. Next, the sections were treated with mouse monoclonal mast cell tryptase antibody and goat polyclonal serotonin antibody and incubated overnight at 4℃. After PB washes, the sections were exposed to Donkey Anti-Mouse IgG Alexa Fluor 488 and Donkey Anti-Goat IgG Alexa Fluor 594 for 2 hours. Subsequently, the sections were washed with PB and stained with DAPI for 6 minutes. After washing with PB, the sections were observed under a confocal laser scanning microscope. Double immunohistochemical staining using the same staining protocol was undertaken, to examine the co-expression of mast cell tryptase and HA.

Proper citation: Ning Ding 2018. Mast cells play an important regulators of acupoint sensitization via the secretion of tryptase, 5-hydroxytryptamine, and histamine. protocols.io https://dx.doi.org/10.17504/protocols.io.mmpc45n Copy   

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