eamag/protocols-with-steps huggingface dataset:
TITLE: Intrinsic water use efficiency estimate: an isotopic method 
AUTHORS: Flavie Gerle, Pauline Malherbe, Christelle Boisselet, David Lafleuriel, Julien Godfroy, Pierre Lochin, Baptiste Marteau, Hervé Piegay, Sara Puijalon, Antoine Vernay

[DESCRIPTION]
Protocol summary and key steps

This protocol describes in detail how to calculate the intrinsic water use efficiency (WUEi) from 13C measurements in phloem samples collected at breast height in trees. Basically, the isotopic signature of photosynthetized carbohydrates (13C/12C) in the phloem sap inform on the assimilation condition: a higher (13C/12C) is associated with a lower transpiration rate, increasing heavy isotope (13C) use by the plant (Dawson et al. 2002). A higher 13C/12C in the phloem content reflect a higher proportion of assimilated C compared to transpiration rate, in other words, a higher WUEi. The method described here is based on the isotopic discrimination of δ13C in trees and considers ecophysiological processes of stomatal and mesophyll conductance (gs and gm, respectively). The protocol aims to provide the operator with a step-by-step method from the collection of phloem samples in the field to the
final calculation. The protocol is already described in the scientific literature (Klein et al. 2016; Seibt et al. 2008; Vernay et al. 2020) but this document addresses more practical and technical details that a practitioner might expect. WUE (water use efficiency) corresponds to plant productivity or the productivity of an organ of the plant regarding the amount of water used to produce this fresh matter (Théroux Rancourt 2014). At an individual scale, WUE considers a ratio of the biomass produced compared to the amount of water used to produce this biomass. At the leaf level, WUEi (intrinsic WUE) determines the ratio of CO2 assimilation per amount of water transpired and can take into account the micro-environment of the leaf, by considering gs/gm to be more precise (Vialet-Chabrand 2013).







[STEPS]
TITLE: rotocol for data collection for  association data between VEMP in the time domain and the frequency domain.
AUTHORS: Aline Tenório Lins Carnaúba, Kelly Andrade, Pedro Menezes

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Protocol for data collection for  association data between VEMP in the time domain and the frequency domain. The protocol has general data fields, radiographic data and VEMP data in the time domain and the frequency domain.</div></div>

[STEPS]
TITLE: Fluoxetine as an Anti-Inflammatory Therapy in SARS-CoV-2 Infection - Protocol v1.1
AUTHORS: Justin Fortune Creeden, Ali Sajid Imami, Hunter M. Eby, Cassidy Gillman, Kathryn N. Becker, Jim Reigle, Elissar Andari, Zhixing K Pan, Sinead M O’Donovan, Robert E McCullumsmith, Cheryl B McCullumsmith

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Hyperinflammatory response caused by infections such as Severe Acute Respiratory SyndromeCoronavirus 2 (SARS-CoV-2) increase organ failure, intensive care unit admission, and mortality. Cytokine storm inpatients with Coronavirus Disease 2019(COVID-19) drives this pattern of poor clinical outcomes and is dependent upon the activity of the transcription factor complex nuclear factor kappa-light-chain-enhancer of activated B cells(NF-</span><span style = ":UNDERLINE;">κ</span><span>B) and its downstream target gene interleukin 6 (IL6). In this study, we compare transcriptomic signatures from a variety of drug-treated or genetically suppressed (i.e.knockdown) cell lines in order to identify a mechanism by which antidepressants such as fluoxetine demonstrate non-serotonergic anti-inflammatory effects. Our results demonstrate a critical role for the IL6 signal transduction protein(IL6ST or gp130) in fluoxetine’s ability to act as a potential therapy for hyperinflammatory states such as asthma and sepsis.</span></div></div>

[STEPS]
?. [Workflow]
Identify Drug SignaturesWe searched iLINCS for the L1000 drug signatures for Fluoxetine, Paroxetine, Bupropion and Dexamethasone.
?. [Workflow]
Download Drug SignaturesWe downloaded all the 436 identified signatures for further processing.
?. [Workflow]
Generating High Confidence SignaturesWe filtered all signatures to keep only the genes that had a Log2 Fold Change value greater than 0.85 or less than -0.85 to generate a high confidence signature
?. [Workflow]
Identifying Concordant SignaturesThe generated high confidence signatures were uploaded to iLINCS and searched against the Consensus Gene Knockdown Signature database for concordance against 27 inflammation related genes.
?. [Workflow]
Cell Line MatchingThe resulting list of concordant signatures was matched with the list of input signatures and only those signatures were kept that had the input and concordant signature in the same cell line. This resulted in 779 total signatures.
?. [Workflow]
Maximising Absolute ConcordanceThe list of 779 total signatures was further simplified by only keeping the match with the maximum absolute concordance score in a given combination of drug, gene and cell line. This reduced the list of signatures to 395.
?. [Workflow]
Summarising ConcordanceThe concordance scores were then further summarised by calculating an arithmetic mean of the concordance scores across multiple cell lines for the same drug-gene combination.
TITLE: MBP Pulldown Assay of ATG9A Truncations
AUTHORS: Adam   Yokom, Xuefeng  Ren

[DESCRIPTION]
MBP Pulldown Assay of ATG9A Truncations

[STEPS]
1. Equilibrate 30 μl of Amylose resin (New England Biolabs, Ipswich, MA). Add >500mL of wash buffer (25 mM HEPES pH 7.5, 150mM NaCl, 1mM MgCl2, 1mM TCEP). Slow spin to pellet resin. ~1000rpm for 1 minute should be good. Repeat X3
2. Add recombinant MBP protien (~1 uM) and ATG13:ATG101 dimer (~3 uM) to Amylose resin
3. Incubate overnight at 4C
4. Wash resin 4x with wash buffer (25 mM HEPES pH 7.5, 150mM NaCl, 1mM MgCl2, 1mM TCEP)
5. Elute samples in 50 uL buffer + 50 mM Maltose
6. Mix eluted samples with lithium dodecylsulfate (LDS)/BME buffer. Heat at 60C for 5 min and run on SDS/PAGE gel
7. Quantify using Fiji ImageJ2
TITLE: Emergency Department Management of AcuteAsthma Exacerbation in Childhood
AUTHORS: Wilson Rocha MD

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Goal: Establish a protocol focusing on the first care of asthma exacerbation in children under 12 years old attending the emergency department of our institution, in the first four hours of initial care. </div><div class = "text-block">Objectives: recognize and classify the severity of asthma exacerbation; administer early treatment with the best possible level of evidence; define criteria for hospitalization and discharge; define criteria for admission to the ICU; develop discharge guidelines to avoid unnecessary returns.</div></div>

[STEPS]
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TITLE: An mHealth app intervention for self-management of knee osteoarthritis: a protocol for a scoping review
AUTHORS: Takashi Kitagawa, Homare Hirokawa, Nanako Otake, Takumi Denda, Kaede Hiraya

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Objective:</span><span> The purpose of this scoping review is to investigate the current status of development and research of mobile health (mHealth) technology or smartphone applications for self-management of knee osteoarthritis (OA) and to summarize their usefulness.</span></div><div class = "text-block"><span style = "font-weight:bold;">Introduction:</span><span> Self-management education for patients with knee OA has been reported to reduce pain, improve activities of daily living, and even reduce healthcare costs. Although there is an increasing number of smartphone apps to support self-management, there have been no comprehensive reviews of the usefulness of them for knee OA.</span></div><div class = "text-block"><span style = "font-weight:bold;">Inclusion/exclusion criteria:</span><span> This review will include patients with knee OA, based on radiography or physician diagnosis. Eligibility criteria will be interventions with apps that have one or more of the following features: record and manage symptoms, provide patient education, and teach and record daily living. Outcomes will be pain, physical function, and quality of life. The study design will be that of an interventional trial or observational study, and should be published in English.</span></div><div class = "text-block"><span style = "font-weight:bold;">Methods:</span><span> PubMed, Web of Science, CINAHL, and CENTRAL will be systematically searched using the keywords “knee osteoarthritis,” “mHealth,” and “self-management” during May 2021. In the first screening step, two independent reviewers will review all of the titles and abstracts to exclude irrelevant articles. In the second screening step, two independent reviewers will review all of the full texts to exclude irrelevant articles. Outcomes will be focused on pain, physical function, and quality of life. Chapters will be divided for each outcome and the results will be described.</span></div></div>

[STEPS]
TITLE:  LAMP - Kit WarmStart Colorimetric LAMP 2x Master Mix (DNA &amp; RNA), marca New England Biolabs
AUTHORS: marcellaa

[DESCRIPTION]
Reação de LAMP para identificação de isolados de Sporothrix brasiliensis - Kit WarmStart Colorimetric LAMP 2x Master Mix (DNA & RNA), marca New England Biolabs.

[STEPS]
SECTION: Preparo
1. Identifique corretamente os eppendorfs com a ID das amostras a serem testadas;
SECTION: Preparo
2. Pipete 5 μL do mix de LAMP em cada eppendorf;


SECTION: Preparo
3. Pipete 1 μL do mix de primers de LAMP;

SECTION: Preparo
4. Pipete 3 μL de água miliq;

SECTION: Preparo
4.1. Nos controles negativos adicione mais 1 μL de água miliq (total de 4 μL);
SECTION: Amostras
5. Pipete 1 μL de amostra em cada eppendorf correspondente; 
SECTION: Aquecimento
6. Incubar os eppendorfs a 65º por 30 minutos;
SECTION: Aquecimento
6.1. Nota: o resultado positivo é percebido pela mudança na coloração do meio de rosa pink para amarelo; mudanças para a cor laranja indicam resultado positivo, necessitando de tempo maior de incubação.

SECTION: Aquecimento
7. Verifique o resultado da reação após 30 minutos; caso não tenha ocorrido nenhuma mudança na coloração, incube por mais 10 minutos e repita o processo até a identificação de alguma mudança na coloração do meio ou até 60 minutos.
TITLE: Bone Decalcification Protocol Using 14% EDTA Buffer Solution pH 7.2 - 7.4
AUTHORS: Donna Peehl, Renuka Sriram, Rosalie Nolley

[DESCRIPTION]
This protocol describes the steps required for successful decalcification.  Decalcification describes the technique for removing minerals from bone or other calcified tissue so that good-quality paraffin sections can be prepared that will preserve all the essential microscopic elements.  Decalcification is carried out after the specimen has been thoroughly fixed and prior to routine processing to paraffin.









[STEPS]
SECTION: Preparation of Ethylenediaminetetraacetic acid (EDTA)
1. Make 14% EDTA solution fresh
a.  Add 140 g free acid EDTA to 700 mL  distilled H2O
b.  On stir plate in the fume hood, add ammonium hydroxide, 30 mL at a time, until solution clears (about 90 mL total)
c.  Add H2O to almost 1 L. Check pH and adjust with ammonium hydroxide dropwise up to pH 7.4, then adjust final volume to1 L

SECTION: Procedure
2. Dissect bone and remove as much soft tissue as possible.
SECTION: Procedure
3. After appropriate fixation in 10% buffered formalin - 1440 min-2880 min hours, wash tissue in distilled H2O or used EDTA solution.

SECTION: Procedure
4. Place tissue in 14% EDTA solution at 4 °C C on a stirring device to circulate the EDTA.  Use enough solution to saturate tissue (fluid volume to tissue ratio is critical for the decalcification process). The EDTA solution should be at least 20X the volume of the tissue to ensure proper decalcification.


 



SECTION: Procedure
5. Periodically check bone for adequate decalcification. Refresh EDTA solution daily for first 5 days, then may leave in same EDTA solution without changing.
SECTION: Procedure
6. Decalcification is complete when bone is soft and pliable. This may take 10 days or more depending on tissue size. Can check by X-ray, or probe with a needle and/or bend tissue to determine if tissue is soft enough to section. Over-decalcification will cause tissue or cells to lose affinity for certain stains.
SECTION: Procedure
7. Rinse once with Phosphate Buffered Saline (PBS) for 30 min minutes


SECTION: Procedure
10. Proceed with tissue processing
SECTION: Procedure
8. Rinse twice in  ddH20 for 30 min minutes

SECTION: Procedure
9. Place in 70% Etanol (EtOH).  Store at  4 °C (no more than 2 weeks)
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p><strong>Background &amp; Aims </strong>Hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) is prevalent worldwide. Despite its limitations, serum alpha-fetoprotein (AFP) remains the most widely-used biomarker for the diagnosis of HCC. This study aimed to assess whether measurement of peripheral plasma Dickkopf-1 (DKK1) and Tie2-expressing monocytes (TEMs) could overcome the limitations of AFP and improve the diagnostic accuracy of HCC.</p>
<p><strong>Methods </strong>Plasma DKK1 level and the percentage of TEMs in peripheral CD14+CD16+ monocytes from HCC patients (<em>n</em>=82), HBV-related liver cirrhosis (LC) patients (<em>n</em>=29), chronic hepatitis B (CHB) infected patients (<em>n</em>=28) and healthy volunteers (<em>n</em>=31) were analyzed by ELISA and flow cytometry. Receiver operating characteristic (ROC) curves were used to analyze a single biomarker, or a combination of two or three biomarkers.</p>
<p><strong>Results </strong>The percentage of TEMs in peripheral CD14+CD16+ monocytes and plasma level of DKK1 in HCC group were significantly higher than those in LC, CHB and healthy control groups (all <em>P</em>-values &lt;0.05). The percentage of TEMs alone was also significantly higher in AFP-negative HCC group than that in LC, CHB and healthy control groups (all <em>P</em>-values &lt;0.05). Plasma DKK1 level alone could not distinguish between AFP-negative HCC and LC patients. ROC curves showed that the optimal diagnostic cutoff value was 550.93 ng/L for DKK1 and 4.95% for TEMs. There was no significant difference in AUC of DKK1, TEMs and AFP in HCC diagnosis between the four groups (all <em>P</em>&gt;0.05). A combination of DKK1, TEMs and AFP measurements increased the AUC for HCC diagnosis as compared with either marker alone (0.833; 95%CI 0.768-0.886). The AUC for TEMs was 0.692 (95% CI 0.564-0.819) in differentiating AFP-negative HCC from LC, with a sensitivity of 80.0% and a specificity of 65.52 %.</p>
<p><strong>Conclusions </strong>TEMs and DKK1 may prove to be potential complementary biomarkers for AFP in the diagnosis of HCC. TEMs rather than DKK1 could serve as a complementary biomarker for AFP in the differential diagnosis of AFP-negative HCC versus LC patients.</p>

[STEPS]
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TITLE: Plate Count Agar
AUTHORS: Andreas Sagen

[DESCRIPTION]
Plate Count Agar (PCA), is a microbiological growth medium to assess viable bacterial growth of a sample.
The total number of living aerobic bacteria can be determined using PCA which is a substrate for bacteria to grow on. The medium contains casein which provides nitrogen, carbon, amino acids, vitamins and minerals to aid in the growth of the organism. Yeast extract is the source for vitamins and glucose is the fermentable carbohydrate. Agar is the solidifying agent. This is a non-selective medium and the bacteria is counted as colony forming units per gram (CFU/g) in solid samples and (CFU/ml) in liquid samples.

[GUIDELINES]
Prepare enough for the necessary number of experiments to perform in order to prevent batch-to-batch variation between experiments. Input chemicals should be of a certain quality in order to prevent contaminants that can impact microbial growth and selection.

[STEPS]
SECTION: 500 mL Plate Count Agar
1. In a bottle, add approx. 400 mL deionized water
SECTION: 500 mL Plate Count Agar
2. Measure and add:
    5 g Tryptone
    2.5 g Yeast extract
    1 g Glucose
    15 g Agar

Materials:
 
 
 
 
SECTION: 500 mL Plate Count Agar
3. Adjust pH to pH 7.0 using sodium hydroxide
SECTION: 500 mL Plate Count Agar
4. Fill bottle to 500 mL total with deionized water
SECTION: 500 mL Plate Count Agar
5. Autoclave liquid at 121 °C for 45 min
SECTION: 500 mL Plate Count Agar
6. Aliquot liquid in 15 mL and 50 mL units and store refrigerated at 4 °C
TITLE: UC Davis - Urea 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">
The enzyme methodology employed in this reagent is based on the reaction first described by Talke and Schubert. To shorten and simplify the assay, the calculations are based on the discovery of Tiffany, et al. that urea concentration is proportional to absorbance change over a fixed time interval. The reaction is monitored by measuring the rate of decrease in absorbance at 340 nm as NADH is converted to NAD.</div></div>

[STEPS]
?. Add 3 μl of calibrator and sample to each well.
?. Add 300 μl of reagent to each well. Incubate at 37°C for 30 seconds. Read at 340 nm.IMPORTANT: Make sure not to add any bubbles to the wells when dispensing reagents, this will interfere with reading in the platereader.
?. Incubate at 37°C for 60 seconds. Read at 340 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: No Title
AUTHORS: 

[DESCRIPTION]
<p>Immunohistochemistry (IHC) is a method that combines biochemical, histological and immunological techniques into a simple but powerful assay for protein detection. IHC provides valuable information as it visualizes the distribution and localization of specific cellular components within cells and in proper tissue context.</p>
<p> </p>
<p>This protocol describes the steps for performing the immunohistochemistry method with paraffin-embedded tissue sections.</p>

[GUIDELINES]
<p>Tissue preparation is a key to successful IHC experiments. Since no universal tissue preparation method will be ideal for all sample and tissue types, the protocol given here is intended as a starting point from which the experimenter must optimize as needed. All conditions should be standardized in order to ensure reproducible results. Keep in mind that you must be careful not to allow tissues to dry out at any time.</p>

[STEPS]
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TITLE: genotyping_PCR
AUTHORS: Ida Barlow

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Protocol for doing PCR on single worm lysate. This is a basic protocol that can be adapted according to specific primers and amplicon size.</div></div>

[STEPS]
?. [Prepare reagents]
Get primers out of freezer or prepare new primers
?. [Prepare reagents]
If primers are ordered new, resuspend lypholised oligo to (== 100 μmol/l). Volume of water (in μl) to add is calculated as Volume =nmol*10Eg. for primer provided as 28.5nmol, add 285 μl of MQ water
?. [Prepare reagents]
If diluting from stock primers, make a a primer mix of 10 μM of each the forward and reverse primer.Eg. +   +  = TOTAL
[water]
[(100  μM) FW primer]
[(100  μM) REV primer]
[primer mix]
?. [Prepare reagents]
Prepare and label PCR tubes
?. [Prepare reagents]
Prepare and label master mix eppendorfs
?. [Prepare reagents]
Get PCR master mix and gDNA out of the freezer. Thaw PCR mix on ice and spin down to collect at the bottom of the tube.
?. [Assemble reagents]
Assemble reagents in master mix tube in following order:1. Water2. Primers3. Taq PCR master mix
?. [PCR program]
Precalculate how many reactions are required for each pair of primers. Remember to include a minus-template control and add 1 to the final number to make sure you don't run out of reagentsEg. for 6 reactions, need to prepare (6+1)X reactions
1X reaction:
?. [Assemble reagents]
Dispense  into each PCR tube
[PCR mix]
?. [Assemble reagents]
Add  to the PCR tube.
[genomic DNA]
?. [Assemble reagents]
Close tubes, make sure the lids are firmly sealed, and flick to ensure all liquid is at the bottom of the tube
?. [PCR program]
Put the PCR tubes in the thermocycler and run with the geno-PCR program
Heat lid:Initial denaturation:35 cycles:1.     2.     3.     Final extension:Final Hold:
TITLE: OMS Atlas OCT Spatial Mapping
AUTHORS: Brett   Johnson, Danielle Galipeau, George Thomas

[DESCRIPTION]
This protocol describes the procedure by which the OMS Atlas serially sections an OCT block, prepares the resulting slides and samples, and then distributes the specimens for downstream analysis.

[BEFORE_START]
Transfer OCT blocks to OHSU Knight Histopathology Shared Resource (HSR) for sectioning and processing.

[STEPS]
SECTION: Preparation
1. Verify the identity of the OCT block to be cut against written request for sectioning.
SECTION: Preparation
2. Remove OCT block from -80 °C freezer and acclimate to cryostat (-20 °C ) for minimum of 180 min.

SECTION: Preparation
3. Label all slides and cryotubes with a unique BEMS ID and Part#, corresponding to the written request and OCT spatial map (below).
 

SECTION: Preparation
4. Prepare PEN membrane slides by exposing close (~15-20cm) to a UV source for 15 min .

SECTION: Sectioning
5. Affix OCT block to cryostat chuck.
SECTION: Sectioning
6. Orient and face block to get adequate amount of core.
Note: Avoid excessive facing to reduce tissue loss.
SECTION: Sectioning
7. Set cryostat to 5 micron sections.
Note: All sections cut from here on should be sequential. The serial order, adjacency, and consistent orientation of the sections are all important factors. Please note any deviations from the protocol.
SECTION: Sectioning
8. Cut first three sections at 5 microns (Part#1-3) and affix onto appropriately labeled slide according to OCT spatial map (step #3 above).
SECTION: Sectioning
9. Change section thickness to 12 microns.
SECTION: Sectioning
10. Cut one section (Part#4) and place in a cryotube.
Note: This is an intermediate section generated when the Cryostat is switching thicknesses. The actual thickness of this section should be about 7µm.
SECTION: Sectioning
11. Cut two sections (Part#5, 6) and place on appropriate membrane slides.
SECTION: Sectioning
12. Change section thickness to 40 microns.
SECTION: Sectioning
13. Cut 2 sections (Part#7) and place both sections in a single cryotube.
SECTION: Sectioning
14. Place all slides, both cryotubes, and remaining OCT block in -80 °C freezer.
Note: No slides are to be fixed under this protocol.

SECTION: Processing
15. Perform hematoxylin and eosin (H&E) staining on slide labeled Part#1 (see OCT spatial map in step #3 above).
SECTION: Processing
16. Deliver unstained slides (Part#2, 3, 5, 6), cryotubes (Part#4, 7), and remainder OCT block (Part#8) to BioLibrary for distribution.
Note: Keep samples frozen at all times. Store at -80 °C. Transfer/ship on dry ice.