eamag/protocols-with-steps huggingface dataset:
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
This protocol outlines the analysis used for intra and interpersonal diversity of the virome and whole metagenome using the Bray Curtis dissimilarity metric. We visualize the differences as bar plots and calculate the statistical significance of the differences using a t-test. Based on the methods from the following publication:<br /><br />Hannigan, Geoffrey D., et al. "The Human Skin Double-Stranded DNA Virome: Topographical and Temporal Diversity, Genetic Enrichment, and Dynamic Associations with the Host Microbiome." <em>mBio</em> 6.5 (2015): e01578-15.<br /><br />

[BEFORE_START]
Supplemental information available at:<br /><br /><a href="https://figshare.com/articles/The_Human_Skin_dsDNA_Virome_Topographical_and_Temporal_Diversity_Genetic_Enrichment_and_Dynamic_Associations_with_the_Host_Microbiome/1281248" target="_blank">https://figshare.com/articles/The_Human_Skin_dsDNA_Virome_Topographical_and_Temporal_Diversity_Genetic_Enrichment_and_Dynamic_Associations_with_the_Host_Microbiome/1281248</a>

[GUIDELINES]
<span class="identifier">sessionInfo</span><span class="paren">(</span><span class="paren">)<br /><br />## R version 3.2.0 (2015-04-16) <br />## Platform: x86_64-apple-darwin13.4.0 (64-bit) <br />## Running under: OS X 10.10.4 (Yosemite) <br />## ## locale: <br />## [1] en_US.UTF-8/en_US.UTF-8/en_US.UTF-8/C/en_US.UTF-8/en_US.UTF-8 <br />## <br />## attached base packages: <br />## [1] stats graphics grDevices utils datasets methods base <br />## <br />## loaded via a namespace (and not attached): <br />## [1] magrittr_1.5    formatR_1.2    tools_3.2.0    htmltools_0.2.6 <br />## [5] yaml_2.1.13    stringi_0.4-1    rmarkdown_0.7    knitr_1.10.5 <br />## [9] stringr_1.0.0    digest_0.6.8    evaluate_0.7</span>

[STEPS]
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TITLE: gRNA design and cloning into Loop L2 plasmids (L2_gRNA-Cas9-CsA and L2_gRNA-CsA plasmids)
AUTHORS: Eftychis Frangedakis, marta tomaselli, Susana Sauret-Gueto

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol explains how to design clone the guide RNA (gRNA) sequence into the L2 plasmids ready to accept the gRNA sequence by cloning with SapI. </div><div class = "text-block">We have two versions of the plasmid, with or without Cas9: the L2_gRNA-Cas9-CsA and L2_gRNA-CsA plasmids. </div><div class = "text-block">With L2_gRNA-Cas9-CsA you transform wild-type sporelings.</div><div class = "text-block">With L2_gRNA-CsA you transform sporelings of a line expressing Cas9.</div></div>

[STEPS]
?. gRNA oligo designOrder two oligos that contain the forward and reverse guide sequence plus the overhangs necessary for ligation (highlighted with bold) into L2_gRNA-CAs9-CsA or L2_gRNA-CsA plasmids: oligo F: 5’-  TCG-NNNNNNNNNNNNNNNNNNNN-gt 3’oligo R: 5’-AAAac-NNNNNNNNNNNNNNNNNNNN-3’Note: Standard de-salted oligos are ok
?. Oligo annealingMix oligos with water as follow:oligo F (100μM)   1μloligo R (100μM) 1μl water  8μl Total volume  10μlAnneal in a thermocycler using the following parameters: 37oC for 30 min, 95oC for 5 min and then ramp down to 25oC at 5oC per min. After annealing the gRNA can be directly cloned into L2_gRNA-CAs9-CsA or L2_gRNA-CsA plasmids without the need of any further processing.
?. Cloning into backbone vector In a 0.2 mL tube set up the following reaction:  AB1  Component
    Volume (μL)
  2  &nbsp;  Sterile water
  53  10x Tango buffer (Thermo Fisher)
  14  &nbsp;&nbsp; 1 mg/mL bovine serum albumin
  (NEB)
    &nbsp;&nbsp; 0.5 
  5  T4 DNA ligase (5 U/µL) (Thermo Fisher)
    &nbsp; 0.25 
  6  10mM ATP (SIGMA)
    &nbsp;&nbsp;&nbsp; 1 
  7  SapI (LguI) (5 U/µL) (Thermo Fisher)
    0.25 
  8L2_393-Csa (25-50 ng)19annealed oligo110  Final volume
  10 Place samples on the thermocycler and incubated using the following program: Assembly:     15 cycles: 3 minutes at 37oC and 4 minutes at 16oC Termination: 5 minutes at 50oC and 10 minutes at 80oC  Transform chemically competent using 1 μL of reaction and plate on LB agar plates with 100 μg/mL spec and X-gal 40. Incubate at 37 oC for 16 h. Confirm with sequencingAssembly:     15 cycles: 3 minutes at 37oC and 4 minutes at 16oC Termination: 5 minutes at 50oC and 10 minutes at 80oC  Transform chemically competent using 1 μL of reaction and plate on LB agar plates with 100 μg/mL spec and X-gal 40. Incubate at 37 oC for 16 h. Confirm with sequencing
AB1  Component
    Volume (μL)
  2  &nbsp;  Sterile water
  53  10x Tango buffer (Thermo Fisher)
  14  &nbsp;&nbsp; 1 mg/mL bovine serum albumin
  (NEB)
    &nbsp;&nbsp; 0.5 
  5  T4 DNA ligase (5 U/µL) (Thermo Fisher)
    &nbsp; 0.25 
  6  10mM ATP (SIGMA)
    &nbsp;&nbsp;&nbsp; 1 
  7  SapI (LguI) (5 U/µL) (Thermo Fisher)
    0.25 
  8L2_393-Csa (25-50 ng)19annealed oligo110  Final volume
  10
TITLE: Generating a low-copy overexpression cell line using PhiC31 integration
AUTHORS: Goran  Tomic

[DESCRIPTION]
This is an in-house adaptation of the commerically available Jump-In system by Thermo. It generates clones with a lower overexpression level than those generated by lentivirial delivery and can be carried out in a standard CL1 tissue culture room. The gene of interest in the attB construct is assembled in-house and is driven by the PGK promoter and contains the BGH pA sequence (assembled from pX28 plasmid (Addgene Plasmid #46850), which had the required sequences). pCMVInt plasmid that expresses the PhiC31 integrase was purchased from Addgene (Plasmid #18935).
This protocol is made combining the protocol for Jump-In system (ThermoFisher) and phiC31 Integrase Vector System (System Biosciences).

[BEFORE_START]
Peform a quick Blasticidin curve to determine the optimal concentration that kills the cells (0-20 ug/mL). Use 24 well plates and try, 0, 2.5, 5, 10, 15, 20 ug/mL Bsd.

[STEPS]
1. Day 1: Use a 6-well plate (0.15x10^6 cells per well). Incubate for 24-48 hours. Transfect if the well is 70-90% confluent.
2. Day 2: Make DNA-Lipofectamine™ 3000 complexes in serum-free medium such as Opti-MEM™ Reduced Serum Medium. 

For a 6 well plate: 2.5 ug of DNA + 125 uL Opti-MEM + 5 uL P3000 reagent (2 uL/ug DNA). Mix PhiC31 integrase plasmid in a 50:1 ratio (w/w) over the donor i.e. 2500 ng of PhiC31 + 50 ng of GOI/attB plasmid
125 uL OptiMEM + 3.75 uL Lipofectamine 3000
Mix 1:1 and incubate for 15 min at RT
Add 250 uL per well to cells in culture medium
3. Day 3: Split the cells into 10 cm plates at 1:10 and 1:20 ratio and add blasticidin. Observe daily and change blasticidin media every 3-4 days until colonies appear.
Validate the expression of the GOI using qPCR, WB, etc.

N.B. To increase the probability of getting single-copy integration of the targeting expression construct, use the amount of vector that results in less than five drug-resistant colonies in the absence of integrase. Using that amount in the presence of integrase should result in >15 colonies.
It is good to have an empty vector control (to generate Bsd-resistant cells, but without GOI).
Important! We observed that in some clones the expression of the GOI can reduce over time. Best to freeze an early passage of the selected single clones after validation, and check the expression of the GOI before any important experiments.
TITLE: Detection of seeded pathology using tyramide amplification
AUTHORS: Bryan_Killinger, Bryan_Killinger

[DESCRIPTION]
This protocol details the detection of seed pathology using tyramide amplification.

[STEPS]
SECTION: Detection of seeded pathology using tyramide amplification
1. Mount the fixed floating 40-micron sections onto gelatin-coated slides and dry at Room temperature 20 min. 

SECTION: Detection of seeded pathology using tyramide amplification
2.  Rehydrate the slides in TBST (refer materials section) and digest with proteinase K (PK, 20 1621) diluted in TBST for 20 minat 37 °C.
4. Place the slides in blocking buffer (TBST, 3% bovine serum albumin, 2% goat serum) for 60 min and then incubate it 60 min at 4 °C in blocking buffer containing anti-PSER129 antibody EP1536Y (Abcam) diluted 1:50,000. 

5. Next day, wash the slides 3 times in TBST and incubate with biotinylated anti-rabbit antibody (Vector Labs) diluted 1:400 in blocking buffer for 60 min.

6. Wash the slides 3 times in TBST and incubate with avidin-biotin complex (ABC) reagent (Vector labs) diluted in blocking buffer for 60 min.

7. Wash the slides twice with borate buffer 0.1 3077 Sodium tetraborate pH 8.5) and incubate in borate buffer containing 0.003% hydrogen peroxide and 5 micromolar (µM) biotinyl tyramide (Sigma-Aldrich) for 30 min.

8. Wash the slides 3 times in TBST and incubate with ABC reagent for 60 min.

9. Heating the slides for 30 min at 80 °C in 20 millimolar (mM) citrate buffer pH 6.0 before ABC reagent can increase detection sensitivity.

10. Wash the slides in TBST and develop using nickel-enhanced 3,3'-Diaminobenzidine DAB as previously described (refer references section).
11. Counterstain the slides with methylgreen (Sigma), dehydrate with graded alcohols, clear with xylenes, and cover the slides using cover slipps with cytoseal 60 (Fisher Scientific). 
12.  Perform the Brightfield microscopy using Nikon A1 laser scanning microscope. 
13. Perform the density analysis including binary masks and region of interest (ROI) analyses using Elements software (Nikon).

3. Fix the slides in 4% paraformaldehyde for 20 min, rinse 3 times in TBST, and incubate with 3% hydrogen peroxide for 30 min to quench endogenous peroxidases.
TITLE: P9 MODIFICACIÓN PROGRAMAS DOCTORADO
AUTHORS: cgarcia

[DESCRIPTION]
Con el objetivo de adaptar la estructura, contenidos y desarrollo de los programas de doctorado de la EIDUCAM, de forma bianual se pondrán en marcha los mecanismos adecuados para llevar a cabo dicha tarea por parte de los directores de los programas.

[STEPS]
SECTION: P9 MODIFICACIÓN PROGRAMA DOCTORADO
1. En el mes de febrero de los años impares, se pondrá en marcha este procedimiento.
SECTION: P9 MODIFICACIÓN PROGRAMA DOCTORADO
2. Teniendo en cuenta el autoinforme y/o informe de seguimiento de cada programa de doctorado realizado por la Comisión de Calidad del Título (CCT) en el mes de marzo anterior, los Directores de los programas elaborarán una propuesta de modificación, debidamente justificada, de la memoria del programa
SECTION: P9 MODIFICACIÓN PROGRAMA DOCTORADO
3. El Director del programa de doctorado presentará en la Secretaría de la EIDUCAM dicho informe de modificación para su evaluación por el Comité de Dirección de la EIDUCAM (15 de Febrero).
SECTION: P9 MODIFICACIÓN PROGRAMA DOCTORADO
4. Evaluación por el Comité de Dirección de la EIDUCAM.
SECTION: P9 MODIFICACIÓN PROGRAMA DOCTORADO
5. Elevación de propuesta a CPA y/o Consejo de Gobierno de la UCAM.
SECTION: P9 MODIFICACIÓN PROGRAMA DOCTORADO
6. Respuesta desde la Secretaría técnica de la EIDUCAM al director del programa de doctorado para la ejecución, o no, de dicha modificación (15 marzo).
SECTION: P9 MODIFICACIÓN PROGRAMA DOCTORADO
7. El director y/o secretario del programa trasladará la resolución a la CCT, y en caso favorable la CCT informará a la Dirección de Calidad de la Universidad de su intención de modificar el Programa (1 abril).
SECTION: P9 MODIFICACIÓN PROGRAMA DOCTORADO
8. La CCT será la encargada de introducir en la herramienta de ANECA las modificaciones del programa.
SECTION: P9 MODIFICACIÓN PROGRAMA DOCTORADO
9. Validación y envío por parte de la Dirección de Calidad de la Universidad de la modificación a la ANECA.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<div title="Page 1">
<div>
<div>
<div>
<p>The current study was to compare the effects of high-intensity interval training (HI) to mild-intensity endurance training (ME), combined with a high-fat diet (HFD) or control diet (CD), on metabolic phenotype and serum corticosterone levels in rats.Although exercise is linked to benefits of metabolic health, it is surprising that few studies have explored the stress responses and interaction of different exercise regimens and HFD feeding, either independently or interactively.It remains unclear whether different exercise regimens lead to similar metabolic and stress responses and adaptations in the presence of an HFD.</p>
</div>
</div>
</div>
</div>

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
The purpose of this protocol is to isolate and characterize viruses that infect <em>P.olseni, P.marinus.</em>

[BEFORE_START]
<div><strong>What you need before you start:</strong></div>
<ul>
<li>UV and sterilize the hood with reagent alcohol. Work with gloves to prevent contaminating the hood.</li>
<li>Your <em>Perkinsus </em>spp. grown to exponential phase</li>
</ul>

[GUIDELINES]
<p>Refs : <br /><br /><em>Fichorova RN, Lee Y, Yamamoto HS, Takagi Y, Hayes GR, Goodman RP, et al. (2012) Endobiont Viruses Sensed by the Human Host – Beyond Conventional Antiparasitic Therapy. PLoS ONE 7(11): e48418. doi:10.1371/journal.pone.0048418<br /><br /></em></p>
<p><em>Bessarab IN, Liu H-W, Ip C-F, Tai J-H (2000) The complete cDNA sequence of a Type II Trichomonas vaginalis virus. Virology 267: 350–359.</em><em> <br /><br /></em></p>
<p><em>Hafiz Ahmed, José A. Fernández-Robledo, and Gerardo R. Vasta (unpublished work) Glycosidases from Perkinsus marinus : Purification and Characterization of ß-D-glucosidase<br /><br /><br /></em></p>

[STEPS]
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TITLE: Proteomic Analysis of Human Whole Lung Tissue Using Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) Acquisition by Data-Independent Acquisition (DIA) on an Orbitrap Eclipse Tribrid Mass Spectrometer
AUTHORS: J Bons, J P Rose, M A Watson, B Schilling

[DESCRIPTION]
Frozen human whole lung tissues were homogenized using a urea-based buffer and bead mill homogenizer to isolate protein. The protein lysate was subjected to tryptic digestion using S-trap Spin columns. The reconstituted peptide elution was desalted with C18 hydrophilic-lipophilic balance (HLB) cartridges. The final reconstituted peptides were diluted with 2% ACN and 0.1% FA. Proteolytic peptide measurement was completed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) acquisition by Data-Independent Acquisition (DIA) on an Orbitrap Eclipse Tribrid mass spectrometer for peptide/protein identification and quantification.

[STEPS]
TITLE:      ELISA for quantification of IL-21 in human serum or plasma.
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>NK cells and CD4+ T lymphocytes make IL-21. It acts on various immune cells of innate and the adaptive immune systems. IL-21 promotes B and T lymphocyte proliferation and differentiation. It enhances NK cell activity. </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-21 coating antibody is adsorbed onto the microwells by incubation overnight at 4°C.
?. Add 50 µl of human serum. Human IL-21 present in the serum sample binds to antibodies adsorbed to 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-21 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-21 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) to each well.
?. Incubate the microwells in the dark for 15 min.
?. A colored product is formed in proportion to the quantity of human IL-21 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-21 standard dilutions and the human IL-21 sample concentration is determined.
?. For better results place the microplate on a microplate shaker in every incubation.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>In this protocol, we used computer animated stimuli for two mate choice tests of <em>Gambusia affinis</em>, such that stimulus pairs differed only by (a) body size and (b) locomotor activity, but not in other morphological or behavioural traits that could affect mate choice decisions.</p>

[BEFORE_START]
<p>We acclimated the fish to laboratory conditions for at least one month before we conducted behavioral experiments and maintained them in groups comprising both sexes at roughly even sex ratios, at densities of around 40 fish per tank, in several aerated and filtered 200-l aquaria under a 12:12 h light/dark regime. Aquaria were well equipped with plants, twigs and stones.</p>
<p>We fed the fish twice a day <em>ad libitum</em> with commercially available flake food, frozen blood worms (chironomid larvae), as well as <em>Artemia salina</em> nauplii and shrimps. In the stock tanks and all experimental tanks, water temperature was kept at 25 ± 1 °C. Water quality was maintained by exchanging half of the water every two weeks, while one tenth of the water was exchanged every day in case of the (smaller) isolation tanks. Aged tap water was used for water changes and throughout the entire experiment.</p>
<p>After acclimation period, we randomly selected adult focal fish from our stock tanks and isolated them, separated by sex, in 96-l tanks 24 hours prior to behavioral tests. To avoid aggressive interactions between individuals and to enable repeated testing of the same individuals, we kept each focal fish separately in 1.5-l transparent perforated plastic bottles, which allowed water exchange with the environment.</p>
<p>A web cam (KC-QB960AK, Keeper, Shenzhen, China) was fixed in a central position approximately 70 cm above the test tank during all behavioral observations, allowing us to remotely observe the focal fish from above. We introduced an air stone connected to an air pump into the test tanks between trials to guarantee well-oxygenated water, and we changed the water every day after a testing session (every 4 trials). Fish were returned to their isolation tanks between subsequent trials.</p>

[STEPS]
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TITLE: Overall protocol for 2D intact proteoform mapping by MALDI imaging
AUTHORS: Kevin J. Zemaitis, Dusan Velickovic, David J Degnan, Mowei Zhou, Ljiljana.PasaTolic

[DESCRIPTION]
Scope:

An overall protocol outlining several nested sub-protocols for the 2D mapping of intact proteoforms from human tissue by MALDI imaging on an UHMR Q Exactive Orbitrap MS.

Expected Outcomes:

Annotated ion images of intact proteoforms ready to be ingested and disseminated.

[STEPS]
SECTION: Sample sectioning and preparation
1. Tissue provided from TMCs are either shipped already cryo-sectioned, or shipped as tissue blocks and the preparation of tissues for MALDI imaging is performed similar to as described elsewhere. Once samples have been sectioned, tissue preparation has been outlined within the following protocol with several nested steps:
 

SECTION: Instrument preparation and operation
2. After tissue preparation the instrument is the optimized, calibrated, and MALDI imaging is performed according to the protocol below:
 

SECTION: Data visualization and annotation
3. Prior to this stage supplemental top-down proteomics by nanoPOTS/microPOTS LCMS/MS is run, once the imaging is completed the image visualization pipeline is run according to the following protocol. Proteoform annotations are then output and manually verified:
TITLE: 01 PCR
AUTHORS: TJUSLS China

[STEPS]
?. Mix the ingredients according to the table belowTemplate 1-30ngSense Primer（10uM） 2.5ulAnti-sense Primer（10uM） 2.5ul5x SuperStar Omni Buffer 5uldNTP（each 2.5mM） 4ulSuperStar Omni DNA Polymerase 1ul10x Enhancer 5ulddH2O up to 50ul
?. Set the PCR instrument Procedure according to the following tablePre-denaturation: 94~98℃  30secDenaturation: 94~98℃  10sec      ⌉Annealing: 50~72℃  30sec            〉    25~35 cyclesExtension: 72℃   30sec/kb            ⌋72℃  10min4℃ ꝏ
TITLE: NEBExpress MBP Fusion and Purification System (NEB #E8201)
AUTHORS: New England  Biolabs

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>The NEBExpress MBP Fusion and Purification System takes advantage of the strong Ptac promoter and the translation initiation signals of maltose binding protein (MBP) to enhance solubility and expression levels of a desired protein in</span><span style = "font-style:italic;">E. coli</span><span>. The resulting product is an MBP fusion protein, which is then purified by affinity chromatography.</span></div><div class = "text-block"><ul style = "list-style-type:disc;"><li style = "counter-reset:ol0;list-style-type:disc;"><span>Reliable</span><span style = "font-style:italic;">E. coli </span><span>expression: substantial yields (up to 100 mg/L)</span></li><li style = "counter-reset:ol0;list-style-type:disc;"><span>Fusion to MBP has been shown to enhance the solubility of proteins expressed in</span><span style = "font-style:italic;">E. coli</span><span style = "vertical-align:super;">(1)</span></li><li style = "counter-reset:ol0;list-style-type:disc;">Two-step purification: amylose elution followed by TEV Protease cleavage and Ni resin isolation results in a highly pure tag-free target protein</li><li style = "counter-reset:ol0;list-style-type:disc;">Gentle elution with maltose; no detergents or harsh denaturants required</li></ul></div></div>

[STEPS]
?. Subclone the gene of interest into the pMAL-c6T vector.
?. Grow cells containing the pMAL-c6T fusion plasmid in LB containing ampicillin and 0.2% glucose to an A600 of ~0.5.
?. Induce by adding IPTG to a final concentration of .
?. Grow for an additional: at  OR  at  OR  –  at  OR at  to .
37 °C
30 °C
30 Room temperature
12 °C
16 °C
?. Harvest the cells and either freeze immediately or resuspend in 25 ml column buffer (CB) per liter of culture.
?. Lyse the cells by freeze-thaw followed by sonication.
?. Clarify the lysate by centrifugation at .
Centrifuge: 20000 33, 20 min
?. Dilute the supernatant (crude extract) by adding  for every 25 ml crude extract.
[cold CB]
?. Load the diluted crude extract on a 15 ml amylose column.
?. Wash the column with ≥ 12 column volumes of CB.
?. Elute the fusion protein with CB containing.
[maltose]