eamag/protocols-with-steps huggingface dataset:
TITLE: Chimeric Protein-LAG and Protein-LG sandwich ELISA
AUTHORS: Angel  Justiz-Vaillant

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This ELISA was used to study the interactions between protein-LAG (PLAG) and protein-LG (SpLG) with different immunoglobulin preparations from mammalian species. </div></div>

[STEPS]
?. This ELISA was used to study the interactions between protein-LAG (PLAG) and protein-LG (SpLG) with different immunoglobulin preparations from mammalian species. The 96 well microtiter plate was coated overnight at 4°C with 2 µg/µl per well of PLAG in carbonate-bicarbonate buffer pH 9.6.
?. The plate was then treated with bovine serum albumin solution and washed 4X with PBS-Tween. 50 µl of immunoglobulins (1 mg/ml) is added and incubated for 1h at room temperature, and the microplate is rewashed 4X with PBS-Tween.
?. Then, 50 µL of peroxidase-labeled SpLG conjugate diluted 1:5000 in PBS-non-fat milk was added to each well and incubated for 1h at RT. The plate was washed 4X with PBS-Tween.
?. Then, 50 µL of o-phenylenediamine solution (4 mg/mL) was added, and the plate was incubated for 15 min at RT in the dark. The reaction was stopped with 50 µL of a 3M H2SO4 solution.
?. The plate was visually assessed for color development and read on a microplate reader at 492 nm. A cut-off point was calculated as the mean of the optical density of the negative controls multiplied by two. The cut-off value was 0.34.
TITLE: BGISEQ-500 （DNBSEQ-G50） Sequencing
AUTHORS: Jie Huang, Xinming Liang, Yuankai Xuan, Chunyu Geng, Yuxiang Li, Haorong Lu, Shoufang Qu, Xianglin Mei, Hongbo Chen, Ting Yu, Nan Sun, Junhua Rao, Jiahao Wang, Wenwei Zhang, Ying Chen, Sha Liao, Hui Jiang, Xin Liu, Zhaopeng Yang, Feng Mu, Shangxian Gao

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"> BGISEQ-500 is a new desktop sequencer developed by BGI. Using DNA nanoball and combinational probe anchor synthesis developed from Complete Genomics™ sequencing technologies, it generates short reads at a large scale. </div></div>

[STEPS]
?. Test the quality of the sequencing library (see protocol for library preparation) by the Qubit® ssDNA Assay Kit and homogenized at 6ng total amounts.
?. Carry out Rolling circle amplification（RCA）for 10 minutes in an 80 ul reaction volume with pure water, buffer and DNB polymerase
?. Add 20 ul DNBs stopping buffer to stop the RCA reaction
?. Check the quality of the DNBs using the Qubit® ssDNA Assay Kit , concentration should be above 10 ng/µL
?. Add 33ul DNBs loading buffer to the DNBs product from the last step, and place it in the BGIDL-50 (the DNBs loading machine)
?. install the sequencing chip and selected the DNBs loading process (Version: sample load 2.0) to load the DNBs.
?. after loading, take the sequencing chip out and install in the BGISEQ-500 （DNBSEQ-G50）sequencing machine
?. load the reagent sequencing kit and open the sequence control software (Version 1.1.0.10003), and select the sequence process Version 1.0.06 and Zebracall process Version 0.5.0.13875 for sequencing.
?. Sequencing is initiated after the sequencing reagents preloaded and sequencing chip was installed, and this process is finished in about 72 hours. When the whole sequencing was finished, the binary file with bases and quality score were converted into FASTQ format with Phred+33 quality score.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>The <strong>GUS reporter system</strong> (<em>GUS</em>: <a href="https://en.wikipedia.org/wiki/%CE%92-glucuronidase" target="_blank">β-glucuronidase</a>) is a <a href="https://en.wikipedia.org/wiki/Reporter_gene" target="_blank">reporter gene</a> system, particularly useful in plant <a href="https://en.wikipedia.org/wiki/Molecular_biology" target="_blank">molecular biology</a><sup class="reference"><a href="https://en.wikipedia.org/wiki/GUS_reporter_system#cite_note-GUS_in_plants-1" target="_blank">[1]</a></sup> and microbiology.<sup class="reference"><a href="https://en.wikipedia.org/wiki/GUS_reporter_system#cite_note-GUS_in_microbes-2" target="_blank">[2]</a></sup>Several kinds of GUS <a href="https://en.wikipedia.org/wiki/Reporter_gene_assay" target="_blank">reporter gene assay</a> are available, depending on the substrate used. The term <strong>GUS staining</strong> refers to the most common of these, a histochemical technique. (Source: https://en.wikipedia.org/wiki/GUS_reporter_system)</p>
<p> </p>

[GUIDELINES]
<ul>
<li>This GUS staining solution recipe is for a volume of 500mL.</li>
<li>The solution can be partitioned into smaller tubes and stored long term at -20<sup>o</sup>C in the dark, as the GUS staining solution is light-sensitive.</li>
</ul>

[STEPS]
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TITLE: U Mass - Energy balance – food intake, energy expenditure, physical activity
AUTHORS: Jason Kim

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary:</span></div><div class = "text-block"><span>The TSE PhenoMaster/LabMaster Metabolic Cage system is used to measure indirect calorimetry, food/water intake, energy expenditure, and physical activity in individually housed awake mice.  The experiment noninvasively measures VO</span><span style = "vertical-align:sub;">²</span><span> consumption and VCO</span><span style = "vertical-align:sub;">² </span><span>production rates in individual mice using metabolic chambers and calculates the respiratory exchange ratio (respiratory quotient) to reflect energy expenditure.  Metabolic cage measurement is conducted continuously for 72 hours (3 days) to account for acclimation of mice housed in home cages during the study.  With our high-speed Siemens O</span><span style = "vertical-align:sub;">²</span><span>/CO</span><span style = "vertical-align:sub;">²</span><span> sensing unit, indirect calorimetry measurements may be performed at a 20-min interval for a full 12-cage study.</span></div></div>

[STEPS]
?. Metabolic cage food and water baskets are calibrated.
?. Drink and food baskets are filled with water and appropriate diet.
?. Corncob bedding is added to the cages.
?. Mice are individually housed in metabolic cages and checked daily for access to food and water.
?. Software is set to collect data at selected interval for 3 consecutive days (72 hour measurement period).
?. Post-run data are exported and analyzed using Microsoft Excel program.
?. Mice should be assessed for body composition (whole body lean mass) prior to metabolic cage measurement in order to incorporate whole body lean mass data for energy expenditure calculation.
?. Metabolic cages require monitoring on a daily basis to ensure food and water are accessible to the mice.
TITLE: Como importar in protocolo existente
AUTHORS: Nastia  Malochka, Gabriel   Gasque

[DESCRIPTION]
1 Send us your protocol.

2 We enter and check your protocol.

Our editorial team will enter the protocol and double check it to make sure there are no errors or typos relative to the document that you provided.

3 Ready for your review.

We will send you the protocol for review.

4 Edit and/or publish at any time.

If all looks good, the protocol will be reassigned to you privately and you can then further edit (add photos/videos/keywords or tweak steps) and decide whether to publish the protocol or share it privately with colleagues whenever you're ready.

[STEPS]
1. Si desea enviar un protocolo existente a protocols.io, visite primero la siguiente pagina de internet: https://www.protocols.io/we-enter-protocols
2. En esta pagina, suba el protocolo que desea que nosotros importemos, a través de la ventana que se indica en la figura y que puede encontrar abajo en la página: 
  

3. Puede adicionar notas, comentarios relevantes o instrucciones especiales para los editors en la ventana "note". Vea figura:
  

4. Ingrese el siguiente código: 4CCF6D0074  

5. Cuando este listo,  envie el protocolo con el botón: CONTINUE .
TITLE: Protocol for the systematic review of  the efficacy of the fixed combination of latanoprost and timolol versus other fixed combinations for primary open-angle glaucoma and ocular hypertension
AUTHORS: Shaohua Huang, Yi Xing, Lijuan Zhu, Ke Zhang

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Background:</span><span>Fixed-combination (FC) therapy is used in primary open-angle glaucoma (POAG) and ocular hypertension (OHT) patients who require more than one medication to reach their target intraocular pressure (IOP). Currently, there are several FC therapies available for the treatment of glaucoma. The FC of latanoprost/timolol (LTFC) is a commonly used. We will conduct a systematic review to compare the IOP-lowering effects of LTFC with other FCs for patients with POAG and OHT.</span></div><div class = "text-block"><span style = "font-weight:bold;">Materials and methods:</span><span> We will search PubMed, EMBASE, the Cochrane Library, and Web of Sciencefor randomized-controlled clinical trials and cross-over studies. The outcomes  are mean IOP and IOP fluctuation after one month of treatment. Meta-analysis will be carried out using RevMan (version 5.1) software. After conducting meta-analyses, we will rate the quality of each meta-analysis as high, moderate, low, or very low using the “GRADE” system. </span></div></div>

[STEPS]
TITLE: Lambda Fague Lysis 
AUTHORS: Laura Sánchez

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Lambda phage is a bacteriophage that infect Gam- bacterias, such as E.coli, trough the outer membrane protein LamB. We use the Lambda phage to characterize if the AEGIS's display system has been properly express.</div></div>

[STEPS]
TITLE: Immune Cell Isolation from Mouse Spleen
AUTHORS: Grace Burgin, Noga Rogel, Moshe Biton

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">To isolate immune cells from a mouse spleen before sorting </div></div>

[STEPS]
?. Prepare 50mL of 10% FBS RPMI media
[FBS]
[RMPI]
**other media may be used
?. Wet both sides of a 70μm filter with media above a 50mL conical (make sure there is some media in the conical)
?. Place spleen on filter, use the plunger base of a syringe to mash the spleen on to the filter while pouring media through
?. Continue pouring media through filter while mashing until spleen is completely mashed. About 30 to 35mL of media should be used
35 ml
?. Keep suspended cells on ice in the 10% FBS RPMI media until preparing for sort
?. Spin cells down at 350g for 3 min (pulse to 550g if necessary)
?. Remove supernatant
?. Resuspend pellet in 1mL of ACK lysing buffer for 1 min on ice
?. Add 30mL of media, spin down at 350g for 3 min (pulse to 550g if necessary)
**if pellet is still red, another round of ACK might be needed
30 ml
?. Stain with desired antibodies, filter with sorting filters, dilute if large pellet
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol was used to generate a single cell suspension from adult human lung tissue. The procedure is carried out on ice, reducing artifact gene expression changes. </p>

[GUIDELINES]
<div>
<p><strong>Enzyme Mixes</strong></p>
<p> </p>
<p><strong>Coll. A/Elastase/Dispase Enzyme Mix (make two tubes - each 1 mL)</strong></p>
<p>60 µL Collagenase A 100 mg/mL – 6 mg/mL final (Sigma, 10103578001)</p>
<p>100 µL elastase 43 u/mL - 4.3 u/mL final (Worthington, LS002292)</p>
<p>100 µL Dispase 90 u/mL – 9 u/mL final (Worthington, LS02100)</p>
<p>5 µL 1 M CaCl2 – 5 mM final</p>
<p>5 µL DNAse (125 U/mL)</p>
<p>730 µL DPBS (no Ca, no Mg)</p>
<p><strong>+13 mg tissue per 1 mL enzyme mix</strong></p>
<p> </p>
<p> </p>
<p> </p>
<p> </p>
</div>
<div>
<div>
<h3>BEFORE STARTING</h3>
<div>
<p>-Prepare enzyme mixes and leave on ice.</p>
<p>-Cool centrifuges to 4 °C.</p>
</div>
</div>
</div>

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
Transformation of heat-shock competent E. coli cells

[BEFORE_START]
For incubation on ice, make sure the tubes are standing in an ice-water mix, because without water, the cooling effect of ice is not reproducible due to the air between the ice fragments, especially if you have to incubate for a certain period of time.

[STEPS]
?. 
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TITLE: The impact of globalization on oral health in Moorea, French Polynesia
AUTHORS: Kenechi Elvis Obiorah

[DESCRIPTION]
    A literature and art review of oral health at the time of European contact and throughout written history and a survey on the oral health quality of native Pacific Islanders today to examine the effects of globalization more specifically the introduction of highly processed carbohydrates and sugars have influenced the oral health of Indigenous Pacific Islanders.

[STEPS]
SECTION: A digital collection of art pieces and literature describing/showing oral health quality of Native Pacific Islanders at early contact time.
3. On a "food-for-thought truck" data will be collected during dental consultations by Tahitian dentists and compiled to access the oral health quality of each patient. Cavities, gum diseases, severe tooth decay/dead teeth, and gum recession are the data points. Each oral health disease will be ranked from 1 to 5. This data will be scaled to a standard oral health quality expected for each participant's age range.
SECTION: Oral health quality and hygiene practices of native Pacific Islanders on Moorea, French Polynesia
1. Art pieces showing oral health quality (full set of teeth) will be compiled into a digital document. A literature review of journals and papers describing oral health quality from missionary/colonists/military accounts during the early contact period in the Pacific will be compiled aswell. Information on pre-contact forms of oral health hygiene practices will also be compiled.
SECTION: Oral health quality and hygiene practices of native Pacific Islanders on Moorea, French Polynesia
2. Older native Pacific Islander participants will be surveyed additionally about the changes they've seen in terms of oral hygiene practices and oral health to further examine globalization's effects on oral health.
SECTION: A digital collection of art pieces and literature describing/showing oral health quality of Native Pacific Islanders at early contact time.
4. Diet information will also be collected, and separated into two major categories, a more traditional diet vs a more "Western diet". Of both diets, food types are to be accessed, foods high in refined sugar, high calcium/vitamin D foods, and foods rich in fluoride.
SECTION: A digital collection of art pieces and literature describing/showing oral health quality of Native Pacific Islanders at early contact time.
5. Then oral hygiene practices will be surveyed, the western standard is to brush at least twice a day and floss at least once. Other oral hygiene practices will be surveyed aswell: the use of chewing sticks, the use of picks, and the use of salt which have all been proven beneficial for oral health.
TITLE: Mannitol Agar
AUTHORS: Adrien Assie, Buck Samuel

[DESCRIPTION]
Mannitol agar recipe

[STEPS]
1. Start with 850mL distilled water
2. 3g Peptone
3. 5g Yeast extract
4. 25g Mannitol
5. Bring to 1000mL distilled water
6. (For LB Agar add 15 g agar)
7. Autoclave
TITLE:  Evaluating large spontaneous deletions in a bovine cell line selected for bovine viral diarrhea virus resistance - MDBK reads mapping to regions in genome  not shared with CRIB cell line 
AUTHORS: aspen.workman  , mike.heaton  , Dennis A. Webster, Gregory P Harhay, Tim  Smith, Shollie    M Falkenberg, Daniel    F. Carlson, Tad S Sonstegard, ted.kalbfleisch  

[DESCRIPTION]
Bovine viral diarrhea virus (BVDV) entry into bovine cells involves attachment of virions to cellular receptors, internalization, and pH-dependent fusion with endosomal membranes. The primary host receptor for BVDV is CD46; however, the complete set of host factors required for virus entry is unknown. The Madin-Darby bovine kidney (MDBK) cell line is susceptible to BVDV infection, while a derivative cell line (CRIB) is resistant at the level of virus entry. We performed complete genome sequencing of each to identify genomic variation underlying the resistant phenotype with the aim of identifying host factors essential for BVDV entry. Three large compound deletions in the BVDV-resistant CRIB cell line were identified and predicted to disrupt the function or expression of the genesPTPN12,GRID2, andRABGAP1L. However, CRISPR/Cas9 mediated knockout of these genes, individually or in combination, in the parental MDBK cell line did not impact virus entry or replication. Therefore, resistance to BVDV in the CRIB cell line is not due to the apparent spontaneous loss ofPTPN12,GRID2, orRABGAP1Lgene function. Identifying the functional cause of BVDV resistance in the CRIB cell line may require more detailed comparisons of the genomes and epigenomes.

[STEPS]
SECTION: Check BAM Files
3. Cell line library BAM files
Illumina libraries of CRIB and MDBK cell lines
The library names are

LIB14393_Bovine_CRIBcells.Bt_ARS-UCD1.2.realigned.bam
LIB14394_Bovine_MDBKcells.Bt_ARS-UCD1.2.realigned.bam

check files 


LIB14393_Bovine_CRIBcells.Bt_ARS-UCD1.2.realigned.bam
 


 



 



LIB14394_Bovine_MDBKcells.Bt_ARS-UCD1.2.realigned.bam
 

 

 

SECTION: Data Conversion
4. Convert BAM to BED


 



 

SECTION: Inputs
1. Required software
 

 

SECTION: Inputs
2. Get RepeatMasked ARS-UCSC2.1 RepeatMasked Bovine Genome features 

Goto http://genome.ucsc.edu/cgi-bin/hgTables to create RepeatMasked ARS-UCSD2.1 bovine genome

  
  

BED file of RepeatMasked Regions of ARS-UCSD bovine genome is   

SECTION: Groom Data
5. Remove features from the MDBK file that intersect with repeats in ARS-UCD1.2.RepeatMask.bed 



 



SECTION: Groom Data
6. Find those features (reads) present in MDBK that do no overlap features (reads) in CRIB

 
Output:
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p><span class="TextRun SCX169215876" lang="EN-US" style="font-size: 14pt; font-family: Times New Roman,serif; line-height: 23px;" xml:lang="EN-US"><span class="NormalTextRun SCX169215876" style="background-color: inherit;">The goal of this protocol is to produce carbon-fiber probes for use with an ICPMS or ESI </span></span><span class="TextRun SCX169215876" lang="EN-US" style="font-size: 14pt; font-family: Times New Roman,serif; line-height: 23px;" xml:lang="EN-US"><span class="SpellingError SCX169215876" style="background-color: inherit;">PrepFast</span></span><span class="TextRun SCX169215876" lang="EN-US" style="font-size: 14pt; font-family: Times New Roman,serif; line-height: 23px;" xml:lang="EN-US"><span class="NormalTextRun SCX169215876" style="background-color: inherit;">/</span></span><span class="TextRun SCX169215876" lang="EN-US" style="font-size: 14pt; font-family: Times New Roman,serif; line-height: 23px;" xml:lang="EN-US"><span class="SpellingError SCX169215876" style="background-color: inherit;">Seafast</span></span><span class="TextRun SCX169215876" lang="EN-US" style="font-size: 14pt; font-family: Times New Roman,serif; line-height: 23px;" xml:lang="EN-US"><span class="NormalTextRun SCX169215876" style="background-color: inherit;">.</span></span><span class="TextRun SCX169215876" lang="EN-US" style="font-size: 14pt; font-family: Times New Roman,serif; line-height: 23px;" xml:lang="EN-US"><span class="NormalTextRun SCX169215876" style="background-color: inherit;"> </span></span></p>

[GUIDELINES]
<p><strong>Supplies:  </strong></p>
<p> </p>
<ul>
<li>Sample tubes (Glass Expansion). NFT2-025 Unifit connector with 1.3 mm OD  x 0.25 mm ID x 2000 mm long sample tube (PKT. 10). You could use the 0.5 mm ID tubes for higher flow rate, but we often prefer the smaller ID tubes for ICPMS work because they have a low dead-volume and thus a quick uptake time. For the Prepfast/SeaFast, it might be better to get some 0.5mm ID tubes.  </li>
<li>Carbon fiber (CST – The Composites Store, Tehachapi, CA). 2.5 mm OD, 1.5 mm ID carbon fiber tubes  </li>
<li>FEP inner end cap material (McMaster). 8703K113, 0.05" before shrinking, FEP, 1.3:1 shrink ratio  </li>
<li>FEP middle end cap material (McMaster). 8703K115, 0.08" before shrinking, FEP, 1.3:1 shrink ratio  </li>
<li>FEP outer sheath material (McMaster). 8703K115, 0.12" before shrinking, FEP, 1.3:1 shrink ratio </li>
</ul>

[STEPS]
?. 
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TITLE: PEI Coating
AUTHORS: kat.titterton  

[DESCRIPTION]
PEI coating 96 and /or 384 wps for highly adherent NGN2 iN replating on DIV3

Based on SOP from Max+ Bobby / Background optimization results

Also refer to: https://www.protocols.io/view/pei-laminin-coating-b5y8q7zw

[STEPS]
SECTION: PEI Stock Prep (1x)
1. Add 500 mg of   to a 50mL conical tube. 
 



SECTION: PEI Stock Prep (1x)
2. Add 30 mL of   to the tube

SECTION: PEI Stock Prep (1x)
4. Add   to bring volume up to 45 mL.  Mix again.

SECTION: PEI Stock Prep (1x)
5. Filter-sterilize: pour concentrated PEI solution into a 500mL filter flask.  

FOR 1x stock final [PEI] =  1 mg/1 mL: Add  to bring volume up to 500mL.

SECTION: PEI Stock Prep (1x)
6. Aliquot PEI solution into 50mL conicals. Use immediately or proceed to step 7

SECTION: PEI Stock Prep (1x)
7. Store in -20 °C freezer #13 shelf #9

SECTION: PEI Stock Prep (1x)
3. Heat the PEI solution t0 ~37 °C on beads. Mix intermittently until PEI is dissolved.
TITLE: Analyzing cellular ATP levels
AUTHORS: Louise Uoselis

[DESCRIPTION]
Protocol for analysis of cellular ATP levels using the Promega Mitochondrial ToxGlo Kit.

[STEPS]
SECTION: Day 1
1. Seed cells into white opaque walled 96 well plates in 80 µL of media/well, aiming for a confluency of ~80-90% the next day at the time of treatment. Fill all surrounding wells with water to prevent evaporation of the experimental samples. Make sure you seed a DMSO treatment control for each sample collection point. 

SECTION: Day 2
2. Treat cells as desired in 80 µL of media/well.

SECTION: Day 2
3. If analysing cells cultured in galactose media, change the media to galactose media 12 – 24 h prior to sample collection. 
SECTION: Day 2
4. To analyse ATP levels, warm the 2x ATP detection reagent to Room temperature, add 80 µL of the 2x ATP detection reagent to each well using a multichannel pipette

SECTION: Day 2
5. Place the plate on a shaker block set to Room temperature, cover the plate in foil, and incubate the plate shaking at   for5 min

SECTION: Day 2
6. Immediately take the plate to a plate reader that can read luminescence signal.