eamag/protocols-with-steps huggingface dataset:
TITLE: HuBMAP | Digestion and scRNA Analysis of Skin
AUTHORS: Arivarasan Karunamurthy, HJ Park

[DESCRIPTION]
This method details digestion and scRNA analysis of the HuBMAP tissue skin specimens.

[STEPS]
1. The fresh skin sample is digested enzymatically using Miltenyi Biotec Whole Skin Dissociation Kit, human for 2 hours and further dispersed using the Miltenyi gentleMACS Octo Dissociator. 
2. The resulting cell suspensions are filtered through 70 µm cell strainers twice and resuspended in phosphate-buffered saline (PBS) containing 0.04% BSA. 
3. Resulting cell suspensions are then loaded into 10× Genomics Chromium instrument (Pleasanton, CA) for library preparation. V1 and V2 single-cell chemistries were used per manufacturer’s protocol. 
4. The resultant libraries are sequenced (~200 million reads/sample), using the Illumina NextSeq-500 platform. 
5. The output sequencing reads were then examined by quality metrics, transcripts mapped to reference human genome (GRCh38) and assigned to individual cells according to cell barcodes, using Cell Ranger (10× Genomics).
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
This is the quick version of the Monarch® Plasmid DNA Miniprep Kit Protocol (NEB #T1010). For the full protocol, please click <a href="https://www.neb.com/protocols/2015/11/20/monarch-plasmid-dna-miniprep-kit-protocol-t1010" target="_blank">here</a>.

[BEFORE_START]
<ul>
<li>For 50-prep kit add 24 ml of ethanol to 6 ml of Monarch Plasmid Wash Buffer 2</li>
<li>For 250-prep kit add 144 ml of ethanol to 36 ml of Monarch Plasmid Wash Buffer 2</li>
</ul>
Add 4 volumes of ethanol (≥ 95%) to one volume of Plasmid Wash Buffer 2.<br /><br />All centrifugation steps should be carried out at 16,000 x g (~13,000 RPM).<br /><br />If precipitate has formed in Lysis Buffer (B2), incubate at 30–37°C, inverting periodically to dissolve.<br /><br />Store Plasmid Neutralization Buffer (B3) at 4°C after opening.

[GUIDELINES]
For detailed protocol and more information, visit <strong><a href="https://www.neb.com/products/t1010-monarch-plasmid-miniprep-kit" target="_blank">www.neb.com/T1010</a></strong><br /><br />The full protocol is available <a href=" https://www.neb.com/protocols/2015/11/20/monarch-plasmid-dna-miniprep-kit-protocol-t1010" target="_blank">here</a>.<br /><br />The video protocol is available <a href="•	https://www.neb.com/tools-and-resources/video-library?videoid=%7B95f48dda-61cc-4b4a-a1cd-582234665960%7D " target="_blank">here</a>.<a href="https://www.neb.com/products/t1010-monarch-plasmid-miniprep-kit" target="_blank"></a>

[STEPS]
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TITLE: Colony PCR (E. coli)
AUTHORS: a.koh

[DESCRIPTION]
Fast and easy PCR to check for cloning inserts. This protocol is not suitable for downstream application of the amplified PCR products. 

[STEPS]
1. Set up 1x PCR buffer mix
5X Green or Colorless GoTaq® Flexi Buffer: 10µl (1x)
PCR Nucleotide Mix, 10mM each: 1µl (0.2mM each dNTP)
upstream primer: 2µl (1.0µM)
downstream primer: 2µl (1.0µM)
GoTaq® G2 Flexi DNA Polymerase (5u/µl): 0.25µl (1.25u)
MgCl2: 2ul (1 mM)
Nuclease-Free Water to: 50µl

Note: Can reduce the final volume to 25µl to save reagents.
2. Using a sterile toothpick or 200µl pipette tips, gently touch a single bacterial colony and dip it into the PCR buffer mix.
3. PCR reaction 
Initial denaturation: 95°C, 2 mins
*Denaturation: 95°C, 1 min
*Annealing: 55°C, 30 sec
*Extension: 73°C, 1 min for every 1kb of amplification
Final extension: 73°C, 5 mins
Storage: 12°C or room temperature depending on when the products will be analysed
*20 to 30 cycles (Generally 20 cycles is more than enough to amplify)

Note: Increasing the annealing temperature will increase the specificity of the PCR reaction.

Important: Run gel electrophoresis immediately as PCR products will normally be degraded due to the presence of DNAse from the lysed E. coli. PCR products not recommended for long term storage.
TITLE: Cerebrovascular Reactivity and Cerebral Autoregulation are Improved in the Supine Posture Compared to Upright in Healthy Men and Women
AUTHORS: Michelle E. Favre, Valerie Lim, Michael J. Falvo, Jorge M. Serrador

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Cerebrovascular reactivity and cerebral autoregulation are two major mechanisms that regulate cerebral blood flow. Both mechanisms are typically assessed in either the supine or seated postures, but the effects of body position and sex differences remain unclear. This study examined the effects of body posture (supine vs. seated vs. standing) on cerebrovascular reactivity during hyper and hypocapnia and on cerebral autoregulation during spontaneous and slow-paced breathing in healthy men and women using transcranial Doppler ultrasonography of the middle cerebral artery. Results indicated significantly improved cerebrovascular reactivity in the supine compared with seated and standing postures (supine= 3.45±0.67, seated= 2.72±0.53, standing= 2.91±0.62%/mmHg, </span><span style = "font-style:italic;">P</span><span><0.0167). Similarly, cerebral autoregulatory measures showed significant improvement in the supine posture during slow-paced breathing. Transfer function measures of gain were significantly decreased and phase significantly increased in the supine posture compared with seated and standing postures (gain: supine= 1.98±0.56, seated= 2.37±0.53, standing= 2.36±0.71%/mmHg; phase: supine= 59.3±21.7, seated= 39.8±12.5, standing= 36.5±9.7°; all </span><span style = "font-style:italic;">P</span><span><0.0167). In contrast, there were no effects of body posture on cerebral autoregulatory measures during spontaneous breathing. Men and women had similar cerebrovascular reactivity and similar cerebral autoregulation during both spontaneous and slow-paced breathing</span><span style = "vertical-align:sub;">.</span><span> These data highlight the importance of making comparisons within the same body position to ensure there is not a confounding effect of posture. </span></div></div>

[STEPS]
TITLE:      ELISA for quantification of IL-28 in human serum.
AUTHORS: Angel  Justiz-Vaillant

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Interleukins (IL) are a type of cytokine first thought to be expressed by leukocytes alone but have later been found to be produced by many other body cells. They play essential roles in the activation and differentiation of immune cells, as well as proliferation, maturation, migration, and adhesion. They also have pro-inflammatory and anti-inflammatory properties. The primary function of interleukins is, therefore, to modulate growth, differentiation, and activation during inflammatory and immune responses. Interleukins consist of a large group of proteins that can elicit many reactions in cells and tissues by binding to high-affinity receptors in cell surfaces. </div><div class = "text-block"><span>Regulatory T-cells synthesize IL-28, which acts on keratinocytes and melanocytes. It stimulates cell presentation of viral antigens to CD8+T lymphocytes. IL-28 also upregulates TLR-2 and TLR-3 expression. IL-28 enhances the keratinocyte capacity to recognize pathogens in the healthy skin. </span><a href="https://www.ncbi.nlm.nih.gov/books/NBK499840" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">[1]</span></a></div><div class = "text-block">Reference</div><div class = "text-block"><span>1. Justiz Vaillant AA, Qurie A. Interleukin. In:</span><span style = "font-style:italic;">StatPearls</span><span>. Treasure Island (FL): StatPearls Publishing; June 12, 2019.</span></div></div>

[STEPS]
?. An anti-human IL-28 coating antibody is adsorbed onto the microwells by incubation overnight at 4°C with carbonate-bicarbonate buffer.
?. Add 50 µl of human serum. Human IL-28 present in the serum sample binds to antibodies adsorbed into the microwells.
?. The microplate is blocked with 3% non-fat milk-PBS buffer and later wash to remove unbound proteins.
?. Fifty (50) µl of biotin-conjugated anti-human IL-28 antibody is added. The optimal dilution must be investigated.
?. The microplate is rewashed with PBS-Tween 20 buffer, pH 7.4.
?. One hundred µl of streptavidin-HRP conjugate is added and it binds to the biotin-conjugated anti-human IL-28 antibody. The optimal dilution of this conjugate must be investigated.
?. The plate is washed following incubation to remove the unbound Streptavidin-HRP.
?. Add 100 µl of 3',3',5',5'- tetramethylbenzidine (TMB; Sigma-Aldrich) into each well.
?. Incubate the microwells in the dark for 15 min.
?. A colored product is formed in proportion to the quantity of human IL-28 present in the sample or standard.
?. The reaction is terminated by addition of 100 µl 3M H2SO4  and the absorbance is measured at 450 nm.
?. A standard curve is made from 7 human IL-28 standard dilutions and the human IL-28 sample concentration is determined.
?. For better results place the microplate on a microplate shaker in every incubation.
TITLE: Sequencing library preparation
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

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol describes the sequencing library preparation for the </span><span style = "font-style:italic;">Tara</span><span> Oceans expedition and is part of </span><a href="https://www.protocols.io/view/viral-to-metazoan-marine-plankton-nucleotide-seque-qv6dw9e" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Viral to metazoan marine plankton nucleotide sequences from the </span><span style = ":;font-style:italic;">Tara</span><span style = ":;"> Oceans expedition</span></a><span>. </span></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/pr-journal/zhnfw26.jpg" /></div><div class = "text-block"><span style = "font-weight:bold;">Figure 1</span><span>: Overview of -omics analysis strategy applied on Tara Oceans samples.</span></div></div>

[STEPS]
TITLE: Region velocity estimation and visulization with seurat
AUTHORS: Chen  Zhang

[DESCRIPTION]
Example pipeline of region velocity estimation and visulization of steady-state model and dynamical model using EM algorithm in R with Seurat to pretreat scRNA-seq data

[STEPS]
SECTION: Read expression matrices  in R
1. 
library(Regionvelocity) data(cell_e_gexp_sper_pb) data(cell_i_gexp_sper_pb) data(cell_RNA_gexp_sper_pb)

SECTION: Alternative step of step 1 to get data from example data of Region velocity package
2. 
library(Regionvelocity) data(cell_e_gexp_sper_pb) data(cell_i_gexp_sper_pb) data(cell_RNA_gexp_sper_pb)

SECTION: Single cell data pretreatment using Seurat
3. 
library(Regionvelocity) data(cell_e_gexp_sper_pb) data(cell_i_gexp_sper_pb) data(cell_RNA_gexp_sper_pb)

SECTION: Single cell data pretreatment using Seurat
4. 
library(Regionvelocity) data(cell_e_gexp_sper_pb) data(cell_i_gexp_sper_pb) data(cell_RNA_gexp_sper_pb)

SECTION: Single cell data pretreatment using Seurat
5. 
library(Regionvelocity) data(cell_e_gexp_sper_pb) data(cell_i_gexp_sper_pb) data(cell_RNA_gexp_sper_pb)

SECTION: Single cell data pretreatment using Seurat
6. 
library(Regionvelocity) data(cell_e_gexp_sper_pb) data(cell_i_gexp_sper_pb) data(cell_RNA_gexp_sper_pb)

SECTION: Region velocity estimation and visulization using steady-state model
7. 
library(Regionvelocity) data(cell_e_gexp_sper_pb) data(cell_i_gexp_sper_pb) data(cell_RNA_gexp_sper_pb)

SECTION: Region velocity estimation and visulization using steady-state model
8. 
library(Regionvelocity) data(cell_e_gexp_sper_pb) data(cell_i_gexp_sper_pb) data(cell_RNA_gexp_sper_pb)

SECTION: Region velocity estimation and visulization using EM algorithm
9. 
library(Regionvelocity) data(cell_e_gexp_sper_pb) data(cell_i_gexp_sper_pb) data(cell_RNA_gexp_sper_pb)
TITLE: UC Davis - Total Cholesterol (TC) Protocol
AUTHORS: Peter Havel

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary:  </span></div><div class = "text-block">Cholesterol esters are enzymatically hydrolysed by cholesterol esterase to cholesterol and free fatty acids. Free cholesterol, including that originally present, is then oxidized by cholesterol oxidase to cholest-4-en-3one and hydrogen peroxide. The hydrogen peroxide combines with HBA and 4-aminoantipyrine to form a chromophore (quinoneimine dye) which may be quantitated at 500-550nm.</div></div>

[STEPS]
?. Add 5 µl of calibrator and sample to each well.
?. Add 300 µl of  reagent to each well. Incubate at 37°C for 5 minutes. Read at 540 nm.
?. Subtract blank readings from final readings. The assay will be linear so the unknown samples can be calculated as (Sample Absorbance ÷ Calibrator Absorbance) × Calibrator Concentration.
TITLE: Mycology media
AUTHORS: Yin-Tse  Huang

[DESCRIPTION]
Mycology media

[STEPS]
1. WA+C agar 
Water agar + chloramphenicol (氯黴素 kill bacteria): As an environmental isolating media

 

2. PDA+C agar
potato dextrose agar + chloramphenicol (氯黴素 kill bacteria): Use when WA+C agar is not working

 

5. Emerson YpSs Agar: For chytrids

 

6. 10% Unclarified V8 Agar
Combine 100 ml V8 juice and 900 ml of distilled water.
Add 1g CaCO3and 0.05 g b-sitosterol and mix well. Leave stir bar in the flask for later mixing.
Add 15g of agar.
Autoclave at 15 psi for 20 minutes.
Stir medium while dispensing to insure good mixing of CaCO3.
20% : 200 ml V8, 800ml d. water, 2g CaCO3, and 0.05g b-sitosterol.
15% : 150 ml V8, 850 ml d. water, 1.5g CaCO3, and 0.05g b-sitosterol.
Reference: Miller, P. M. 1955. V-8 juice agar as a general purpose medium for fungi and bacteria. Phytopathology 45:461-462.
7. SceSel+ medium (Kaltseis et al.,2009)
For isolation of Scedosporium
 

3. quarter-strength PDA  
1. Less nutrient than regular PDA (1/4)
2. For beetle-associated fungi isolation

 

4. MEA (malt extraction agar)
For isolate subculture
TITLE: Immunohistochemistry Protocol for Beta Amyloid Products using USA Detection Kit
AUTHORS: Sam Li

[DESCRIPTION]
<div class = "text-blocks"></div>

[STEPS]
?. Clear Slides: Remove paraffin and hydrate the tissueNote: If using frozen sections, allow slides to come to room temperature for 15 minutes & proceed to step (F) only A. Xylene - 5 minutes in each of (3) different 250 mL containersB. 100% alcohol - 5 minutes in each of (3) different 250 mL containersC. 95% alcohol - 3 minutes in (1) 250 mL containerD. 70% alcohol - 3 minutes in (1) 250 mL containerE. Water - 1 minutes in each of (3) different 250 mL containersF. H2O2 (3%) - 15 minutes in (1) 250 mL container
?. Rinse slides with lab grade water.Note: Lab grade filtered water such as injection grade, cell culture grade, Reverse Osmosis De-Ionization (RODI).
?. Antigen Retrieval (refer to product datasheet, not always required)
?. Apply serum block for at least 5 minutes. Do not wash after this step.
?. Blot off serum block.
?. Apply primary antibody - dilute to 1 µg/mL in PBS.
?. Incubate primary antibody 60 minutes at room temperature.
?. Rinse slides with 1X PBS.
?. Apply USA Linking reagent - 20 minutes incubation.
?. Rinse slides with 1X PBS.
?. Apply Labeling Reagent - 20 minutes incubation.
?. Rinse with 1X PBS.
?. Apply chromogen - 5 minutes incubation. Dilute according to manufacturer's instructions.
?. Rinse slides with lab grade water.
?. Counterstain
?. Clear slides: Dehydrate the tissue.
?. Cover slip slide using Permanent Aqueous Mounting Medium.Note: Do not use xylene based mount with AEC Chromogen as it will dissolve the chromogen.
?. 70% Formic Acid - incubate the slides for 20 minutes at room temperature.Note: This antigen retrieval step is harsh on the tissue. If using frozen sections reduce time to 5-10 minutes or omit if tissue falls off the slide.
?. Rinse Slides with 1X PBS.
?. AEC Chromogen: 20 µL AEC chromogen + 1 mL AEC substrate buffer.
?. DAB Chromogen: 40 µL DAB chromogen + 1 mL DAB substrate buffer.
?. Submerge slides in Mayer’s Hematoxylin for 30 seconds.
?. Rinse under running lab grade water for 1 minute or until water is clear.
?. Submerge slides in Bluing Reagent for 1 minute.
?. Rinse under running lab grade water for 1 minute.
?. 95% alcohol 3 minutes in (1) 250 mL container.
?. 100% alcohol 5 minutes in each of (3) different 250 mL container.
?. Xylene 5 minutes in each of (3) different 250 mL container.
TITLE:       ELISA for quantification of IL-39 in human serum.
AUTHORS: Angel  Justiz-Vaillant

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Interleukins (IL) are a type of cytokine first thought to be expressed by leukocytes alone but have later been found to be produced by many other body cells. They play essential roles in the activation and differentiation of immune cells, as well as proliferation, maturation, migration, and adhesion. They also have pro-inflammatory and anti-inflammatory properties. The primary function of interleukins is, therefore, to modulate growth, differentiation, and activation during inflammatory and immune responses. Interleukins consist of a large group of proteins that can elicit many reactions in cells and tissues by binding to high-affinity receptors in cell surfaces. </div><div class = "text-block"><span>B lymphocytes mainly produce IL-39. It acts on neutrophils inducing their differentiation or expansion. </span><a href="https://www.ncbi.nlm.nih.gov/books/NBK499840" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">[1]</span></a></div><div class = "text-block">Reference</div><div class = "text-block"><span>1. Justiz Vaillant AA, Qurie A. Interleukin. In:</span><span style = "font-style:italic;">StatPearls</span><span>. Treasure Island (FL): StatPearls Publishing; June 12, 2019.</span></div></div>

[STEPS]
?. An anti-human IL-39 coating antibody is adsorbed onto the microwells by incubation overnight at 4°C with carbonate-bicarbonate buffer.
?. Add 50 µl of human serum. Human IL-39 present in the serum sample binds to antibodies adsorbed into the microwells.
?. The microplate is blocked with 3% non-fat milk-PBS buffer and later wash to remove unbound proteins.
?. Fifty (50) µl of biotin-conjugated anti-human IL-39 antibody is added. The optimal dilution must be investigated.
?. The microplate is rewashed with PBS-Tween 20 buffer, pH 7.4.
?. One hundred µl of streptavidin-HRP conjugate is added and it binds to the biotin-conjugated anti-human IL-39 antibody. The optimal dilution of this conjugate must be investigated.
?. The plate is washed following incubation to remove the unbound Streptavidin-HRP.
?. Add 100 µl of 3',3',5',5'- tetramethylbenzidine (TMB; Sigma-Aldrich) into each well.
?. Incubate the microwells in the dark for 15 min.
?. A colored product is formed in proportion to the quantity of human IL-39 present in the sample or standard.
?. The reaction is terminated by addition of 100 µl 3M H2SO4  and the absorbance is measured at 450 nm.
?. A standard curve is made from 7 human IL-39 standard dilutions and the human IL-39 sample concentration is determined.
?. For better results place the microplate on a microplate shaker in every incubation.
TITLE: Media for Fungal Culturing
AUTHORS: Jiri Hulcr, You Li, Sawyer Adams, Demian F Gomez

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes the different media for fungal culturing.</div><div class = "text-block"><span>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 </span><a href="https://doi.org/10.1007/s13199-020-00686-9" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://doi.org/10.1007/s13199-020-00686-9</span></a><span>.</span></div></div>

[STEPS]
TITLE: Title:  Ultrasound Use and its Impact on Clinical Decisions in the Accident and Emergency Department at Georgetown Public Hospital Corporation
AUTHORS: Davendra Kissoon, Sri Devi Jagjit, Jordan Rupp, Brian Bales, Jeremy Boyd

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;font-style:italic;">Introduction</span><span style = "font-weight:bold;font-style:italic;">:</span></div><div class = "text-block">Point-of-care ultrasound (POCUS) is physician-performed at the bedside, and it is a powerful diagnostic tool, especially in resource-limited emergency medicine healthcare settings.This study aims to quantify both the use of ultrasound and its impact on patient care at the Accident and Emergency Department at the Georgetown Public Hospital Corporation.</div><div class = "text-block"><span style = "font-weight:bold;font-style:italic;">Methods</span><span style = "font-weight:bold;font-style:italic;">:</span></div><div class = "text-block">This is a descriptive study of data collected for quality assessment in the GPHC Accident and Emergency Department.Over the course of two months, physicians were asked to record each ultrasound exam performed and record whether the ultrasound results changed patient disposition or the medication used in management.</div><div class = "text-block"><span style = "font-weight:bold;font-style:italic;">Results</span><span style = "font-weight:bold;font-style:italic;">:</span></div><div class = "text-block">During the study period, there were 173 ultrasound data sheets collected representing 426 ultrasound studies.196 studies were positive with pathologic findings (46.0%).The use of ultrasound in patient care either affected the patient’s final disposition or medication 78.6% of the time.</div><div class = "text-block"><span style = "font-weight:bold;font-style:italic;">Conclusion</span><span style = "font-weight:bold;font-style:italic;">:</span></div><div class = "text-block">Ultrasound is used frequently at the Georgetown Public Hospital Corporation for a wide variety of applications.When utilized, POCUS frequently influenced patient care.</div></div>

[STEPS]