eamag/protocols-with-steps huggingface dataset:
TITLE: Crystallisation of SARS-CoV-2 Mpro
AUTHORS: blake.h.balcomb, Peter Marples, Lizbé Koekemoer, Daren Fearon, Charlie Tomlinson

[DESCRIPTION]
The COVID-19 pandemic has highlighted the need to identify novel therapeutic interventions and strategies for pandemic preparedness against Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). This protocol outlines the crystallization protocol and buffer conditions used to obtain reproducible SARS-CoV-2 Mpro crystals suitable for XChem fragment screening.

[STEPS]
SECTION: Crystallisation experiment
4. Protein and buffer requirements:
43.2 µL5 mg/mL  
1.92 mL 
14.4 µL   seeds, dilution 1:250

SECTION: Crystallisation experiment
6. Dispense 20 µL into SwissCI 3 lens plate reservoir wells using a 100 µl multi-channel pipette.
Dispense 150 nL5 mg/mL    to each lens using the SPT mosquito.
Dispense  150 nL to each lens using the SPT mosquito.
Dispense  50 nL to each lens using the SPT mosquito.

Drop ratio: 3:3:1 ratio (150 nl   : 150 nl reservoir solution: 50 nl seeds)
Final drop volume: 350 nl
SECTION: Crystallisation experiment
5. Crystallisation screen composition:
0.1 Molarity (M)  MES pH 6.7 
11% w/v PEG 4000
5% DMSO

Stock solutions used:
1 Molarity (M)  MES adjusted to pH 6.7 with HCl
50% w/v PEG 4000
99.9% DMSO

 

SECTION: Crystallisation experiment
8. Crystal form after ~24 h.
 


SECTION: Crystallisation experiment
7. Incubate at 20 °C for 1440 min in Formulatrix Rock Imager.

Imaging Schedule: The first images are taken after 12 h and the imaging schedule follows a Fibonacci sequence of days for further collections. 
SECTION: Equipment needed
2. Formulatrix Rock Imager (or incubator of choice)
SPT mosquito
 
P100 8 multi-channel pipette
SwissCI 3 lens plate

SECTION: Crystallisation experiment
3. Prepare seed stock:
 
1: 250 dilution    seeds
SECTION: Data collection at Synchrotron
9. Diamond Light Source
Unattended Data Collection (UDC)
Data Collection Temperature: 100K
Detector: DECTRIS EIGER2 X 9M
Beamline: I04-1
Wavelength:  0.9212 Å
Resolution (Å): 1.78
Beam Size (μm): 60 X 50
Number of images: 3600
Oscillation: 0.10°
Exposure (s): 0.0020
Transmission (%): 100
Flux (ph/s): 9.50e+11
SECTION: SARS-CoV-2 Mpro expression and purification
1. The protein used for crystallisation was expressed and purified using the following protocol.
TITLE: Dechorionation of zebrafish embryos with Pronase for metronidazole-mediated ß-cell ablation
AUTHORS: Sandra Rieger

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary: </span></div><div class = "text-block"><span>Zebrafish embryos develop in their eggshell (the chorion) until hatching (~48-72 hours post fertilization). Because the chorion is impermeable to water, it needs to be removed prior to metronidazole treatment and β-cell ablation in Tg(</span><span style = "font-style:italic;">ins:NTR-mCherry</span><span>) transgenic zebrafish embryos. This can be achieved either with forceps or by enzymatic removal using Pronase. The earliest time of removal should be after completion of gastrulation to avoid bursting of the yolk and potential infections.</span></div><div class = "text-block"><span style = "font-weight:bold;">Diabetic Complication:</span></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/pr-journal/bhji4kcn.jpg" /></div></div>

[STEPS]
?. [Potential Pitfalls:]
1. The embryos do not survive after treatment: This could be if embryos were incubated too long in Pronase. Decrease the incubation time and increase the number of washes.2. Dechorionation does not work: Pronase might be too old or animals are older than 24 hours post fertilization. Increase incubation time in Pronase until chorions become soft. This can be up to 10 minutes in 48 hpf larvae.
?. [Protocol:]
For dechorionation, add 9 ml of embryo medium containing 0.003% PTU to 15 ml Falcon tubes containing 1 ml of the Pronase stock solution to make a 10 ml working solution of 1 mg/ml. Pour the working solution into a small petri dish and place the embryos still in their chorion into the solution. Swirl the embryos until the chorion becomes soft (tear off soft chorion with forceps under a stereomicroscope, typically after 3-5 minutes at 24 hours post fertilization). Immediately transfer the embryos into a petri dish with fresh embryo medium. Wash three times in fresh embryo medium.
TITLE: Untitled
AUTHORS: Cui Jian

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

[STEPS]
?. [1]
0 µl
0 µl
0 µl
?. [1]
{"blocks":[{"key":"8v8id","text":"The growing of microbrewerys in Brazil has imposing an increase in the production of different supplies for brewing, iincluding yeast biomass for beer fermentation. In Brazil,only four companies produce and sell yeasts for microbreweries, and only two companies are able to attend microbreweries. Additionaly, these companies should be able to deliver yeast cells to different states and cities in Brazil. During transport, the yeast cells are exposed to thermical stress (due to the lack of temperature control) and chronological aging (due to the long period of storage and transport). When delivered to the microbreweries, those yeast cells display problems during beer fermentation as the consequence of a loweryeast vitality and viability and affecting beer\u2019s quality. Thus, in this work a new colorimetric assay was developed to access the vitality of yeast cells by using resazurin. Resazurin is a highly dichromatic dye, showing a colorimetric change proportional to the yeast cellvitality that can be easily visualized by naked eye. To evalute the potential of resazurin to differentiate yeast cells with low and high vitality, different yeast ale and lager strains (US-05 and W34\/70, respectively) were submitted to heat shock stress and their vitality was accessed by resazurin, testing different variables as concentration of work solution, cellular density and incubation time. The resazurin data, supported by acidification power test, showed a vitality decrese in cells submited to heat shock stress. Futhermore, the sensibility of the resazurin assay was explored during chronological aging of yeast cells. The assay was capable to detect differences of vitality and viability between yeast strains. In addition, the potencial of the resazurin test to predict the fermentative capacity of stressed and non-estressed yeast cells was observed in wort attenuation assays, comfirming the aplicability of resazurin in the microbrewery context.","type":"unstyled","depth":0,"inlineStyleRanges":[],"entityRanges":[],"data":[]}],"entityMap":[]}
?. [1]
?. [1]
TITLE: Pole Test
AUTHORS: Sabina Marciano, Tae-In Kam, Roberta Marongiu, Ted Dawson

[DESCRIPTION]
This behavior test is used to assess motor coordination in mice.

[GUIDELINES]
Main outcome measures: average time to turn, average time to reach the bottom

[STEPS]
1. Place the mouse on the vertical pole with their head oriented upwards 7.5cm from the top of the pole.
2. During day 1, gently guide the mouse to turn around and go to the bottom of the pole. They are trained 3 times.
3. On day 2, they are trained 3 times again without guidance.
4. On day 3, the test day, mice are again placed on the pole 3 times. 
Record the time to reorient to face down and the time to reach the bottom of the pole during each of the 3 trials.
TITLE: Bioconjugation Strategies
AUTHORS: BOC      Sciences

[DESCRIPTION]
Bioconjugation is a chemical technique that is used to couple two molecules together, at least one of which is a biomolecule, such as carbohydrate, nucleic acid, or protein. Functionalization of polymers can provide improved stability, solubility, trafficking pathways, and therapeutic potential for already useful biomolecules in a variety of applications. For instance, synthetically modified polymers can act as a tracker to record changes in cell behavior, as a marker for bio-imaging, or as a carrier to deliver drugs to targeted cells. Apart from that, polymers can also reveal enzyme function and determine protein biodistribution in the biomedical field after linking specific biomolecules.

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p><strong>Fluorescent staining for Callose of sieve tubes of phloem.</strong></p>
<p>Calose (β-1→3-glucan) is the distinguished polysaccharide present on the sieve plates, of each sieve tube member of phloem tissue.  Aniline blue stain specifically callose and it is use as a marker for identification of sieve elements of phloem.</p>
<p> </p>
<p><strong>PROCEDURE – STAINING FOR CALLOSE WITH ANILINE BLUE (modification of Klaus Herburger and Andreas Holzinger (2016).</strong></p>
<p><strong> </strong></p>
<p>Prepare Sørensen’s phosphate buffer (0.1 M, pH=8.0) according with Ruzin (1999).  </p>
<p><strong>Stock solutions. </strong></p>
<ol>
<li>A) 0.2 M of NaH2PO4 (Sodium phosphate monobasic anhydrous, CAS Number: 7558-80-7) BioUltra, ≥99.0% (T) (Sigma-Aldrich S3139).</li>
<li>B) 0.2 M of Na2HPO4 (Sodium phosphate dibasic anhydrous, CAS Number: 7558-79-4) Ultra ≥99.5%, (Sigma-Aldrich S7907).</li>
</ol>
<p><em>To prepare 100 mL of the buffer (0.1 M, pH=8.0), mix 2.7 mL of stock solution (A) with 47.3 mL of stock solution (B) and adjust volume to 100 mL  in a Schott flask.</em></p>
<p>Add 0.5 % Aniline blue (w/v) to freshly prepared Sørensen’s phosphate buffer.</p>
<p> </p>
<ul>
<li>Fix tissue in 2.5% glutaraldehyde in phosphate buffer Sorensen's solution (0.1 M, pH 7.2) and processed for LR white resin embedding according with manufactures.</li>
<li>Obtain transverse semi thin sections (1 µm) with an ultramicrotome and bond the sections to microscope grass slides with heat (60 C) during 15 min.</li>
<li>Stain sections with 0.5 % of Aniline blue (CAS Number: 28631-66-5, Sigma Aldrich, Germany) in Sørensen’s phosphate buffer (0.1 M, pH=8.0) for 1 h under low light intensities (~5 μmol photons m<sup>-2</sup> s<sup>-1</sup>) to prevent degradation of the dye.</li>
<li>Observe tissue under an Epifluorescence Microscope (Axioscope 2 Plus, Carl Zeiss AG) using a Set 01 (excitation: band pass (BP) 365/12 nm; emission: long pass (LP) 397 nm) and connected to an Axiocam MRc5 camera. Calose of sieve elements stain bright green yellow. </li>
</ul>
<p> </p>
<p><strong>References</strong></p>
<p>Herburger, K., &amp; Holzinger, A. (2016). Aniline blue and Calcofluor white staining of callose and cellulose in the streptophyte green algae Zygnema and Klebsormidium. Bio-Protocol, 6(20), e1969. http://doi.org/10.21769/BioProtoc.1969</p>

[STEPS]
TITLE: SUPER rapid kit
AUTHORS: Hyeon Jin Kim, Seong T. Hong

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">We developed a supersensitive capillary reaction kit intended for the direct and qualitative detection of SARS-CoV-2 virus in saliva and nasal swabs. It enables sensitive and specific detection of the SARS-CoV-2 spike antigens in respiratory specimens more accurately than most PCR method within 5 minutes. Our rapid capillary kit will help not only fight but also end COVID-19 by providing the first sensitive self-diagnosis at home as well as POC clinical diagnosis at the health care facilities. </div></div>

[STEPS]
?. Prepare the specimen by collecting saliva, sputum, or nasal swab for testing
?. Dispense 2~5 drops of the specimen into the sampling tube (provided) filled with a general transfer pipette
?. Mix the content by pipetting up & down gently
?. Dispense 5 drops of mixed samples into the sample well of the cassette (provided).
?. Observe the appearance of the blue line for 1 min and record the result.
?. To prepare a specimen solution with a nasal swab, insert the testing swab into the sampling tube, roll along the side of the tube 5 times, and discard the used swab to biohazard waste.
TITLE: CODA: shorthand for calling functions | HuBMAP | JHU-TMC
AUTHORS: Kyu Sang Han, Pei-Hsun Wu, Joel Sunshine, Ashley Kiemen, Sashank Reddy, Denis Wirtz

[DESCRIPTION]
To downsample ndpi or svs images to 10x, 5x, and 1x tifs, use this function:          
      create_downsampled_tif_images
or try Openslide in python
 
To calculate registration on the low resolution (1x) images 
1. calculate the tissue area and background pixels using this function:
      calculate_tissue_ws
2. calculate the registration transforms:
     calculate_image_registration
 
To build a 3D tissue volume using sematic segmentation:
1. generate manual annotations in Aperio imagescope
2. apply the deep learning function to train a model and segment the high resolution (5x or 10x) images:
     train_image_segmentation

To apply the registration to segmented images:
                             apply_image_registration

To build a 3D tissue matrix from registered, classified images:
                             build_tissue_volume
 
To build a 3D cell volume containing nuclear coordinates:
1. Build a mosaic image containing regions of many whole-slide images for cell detection optimization:
                             make_cell_detection_mosaic
2. Manually annotate the mosaic image to get the ‘ground-truth’ number of cell nuclei:
                             manual_cell_count
3. Determine cell detection parameters using the manual annotations on the mosaic image:
                             get_nuclear_detection_parameters
4. Deconvolve the high-resolution (5x or 10x) H&E images before applying the cell detection algorithm:
                             deconvolve_histological_images
5. Detect cells on the hematoxylin channel of the high-resolution images:
                             cell_detection
6. Apply the registration to the cell coordinates:
                             register_cell_coordinates
7. Build a 3D cell coordinate matrix corresponding to the 3D tissue matrix:
                             build_cell_volume

[STEPS]
SECTION: shorthand in the abstract
1. Use the above shorthand to facilitate your workflow by using it as a "cheat sheet"
TITLE: RNA extractions from de-etiolated Arabidopsis seedlings using CTAB
AUTHORS: Akila Wijerathna-Yapa, Andrew Bowerman, Diep Ganguly

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">A CTAB based method for extracting RNA, which is particularly useful for tough tissues. This has been adapted from a protocol that was originally used for pine tree tissue, which is difficult due to the high concentrations of polysaccharides, phenolics, and RNase (Chang, S., Puryear, J. & Cairney, J. Plant Mol Biol Rep (1993) 11: 113. https://doi.org/10.1007/BF02670468). The protocol described herein was an effective alternative to TRIzol based extractions for recovering RNA from juvenile de-etiolated tissues.</div></div>

[STEPS]
?. Harvest tissues into liquid nitrogen and grind (under liquid nitrogen) using mortar and pestle to obtain a fine powder. Ground tissue should be kept in safe-lock tubes in liquid nitrogen (or returned to -80C storage) until all samples are processed. This step is critical for efficient extractions so take your time here.
?. Add 1 mL of prewarmed (65°C) extraction buffer to each tube, mix well and incubate for 5 mins at 65°C (can be longer if needed e.g. 10-15 mins)
?. Add 200 ul of chloroform:IAA (24:1), mix well and spin @ 14,000 rcf for 10 mins. Remove upper aqueous phase to a new tube. Make sure not to disturb or pipette any material from the interface. Repeat chloroform:IAA step twice, being increasingly conservative when recovering the aqueous phase.
?. Add equal volume of 5 M LiCl to aqueous layer, mix well and incubate overnight @ -20°C.
?. Spin tubes @ 14,000 rcf for 20 mins @ 4°C.
?. Remove supernatant with pipette, add 1 mL 80% ethanol and invert tube ~10X. Centrifuge @ 7,500 rcf for 5 minutes @ RT. Remove ethanol and repeat.
?. Remove as much ethanol as possible using a pipette. Air-dry tubes with lid open for 1 min. Resuspend in 30 - 50 ul (depending on expected concentration and purpose, e.g. we aim for ~ 500 ng / ul) DEPC-treated water or low EDTA TE buffer (0.1 mM EDTA, 10 mM Tris base, pH 6.5).
?. Check quality of RNA e.g. visualize ~50 ng RNA on a 1% agarose gel or use BioAnalyser / LabChip GXII.
TITLE: Universal DNA isolation protocol
AUTHORS: Ruslan Kalendar

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The isolation of nucleic acids from a sample is an important step for many molecular biological applications and medical diagnostic assays. This protocol describes an efficient method for purification or/and isolation of nucleic acids from difficult animal tissues, plant material and other samples from which DNA extraction is generally regarded as being difficult which can contain impurities and inhibitors or interfering substances. This method is established acidic CTAB (with a pH value of 5 to 6.8) based extraction protocol that allows for reliable isolation of high molecular weight genomic DNA for removing contaminants from nucleic acids in a sample, e.g., environmental or biological samples such as soil, food, plant, animal or microorganism. DNA may quickly be extracted from samples without phenol, guanidine thiocyanate or 2-mercaptoethanol.</div></div>

[STEPS]
?. In 2 ml tube with mechanically disrupted seeds/leaves/herbarium or DNA solution (CTAB purification) add 1 ml CTAB solution buffer with RNAse A (the sample mass should not exceed 100 mg), vortex very well and incubate the samples at 60-65°C during 60-120 min or longer (long incubation increases DNA yield).
?. Centrifuge at maximum speed in a microcentrifuge for 2-5 minutes. A whitish DNA pellet should be visible.
?. Spin at maximum speed in a microcentrifuge for 2 minutes, transferred the upper aqueous layer to a new 2 ml microcentrifuge tube.
?. Spin at maximum speed in a microcentrifuge for 2 minutes.
?. Transfer the entire clarified upper aqueous layer to a new 2 ml microcentrifuge tube which contains an equal or half volume of 2-propanol and vortex thoroughly.
?. Discard supernatant and wash the pellet by adding 1.8 ml 70% ethanol, vortex thoroughly. At this stage, DNA samples can be stored at room temperature or refrigerated.
?. Centrifuge at maximum speed for 2-5 min and carefully discard the supernatant by decanting or with a micropipette. A whitish DNA pellet should be visible during discarding a supernatant.
?. Transfer theentire clarified supernatant to a new 2 ml microcentrifuge tube contains an equal volume of chloroform. Mix well for 3-5 minutes in the MM300 Mixer Mill at 30 Hz.
?. The DNA pellet does not dry and dissolved immediately in 300 μl 1xTE, pH 8.0 at 55°C for 10-20 minutes.
?. Eppendorf Safe-Lock microcentrifuge tube with tissue sample and glass ball (6 mm) freeze at -80°C, grind in the MM300 Mixer Mill for 2 min at 30 Hz. Alternatively, grind the sample in lysis solution.
TITLE: Human embryonic gonad dissociation with Trypsin-EDTA
AUTHORS: Regina Hoo, Roser Vento-Tormo, Carmen Sancho

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is for enrichment of fetal gonadal cells</div></div>

[STEPS]
?. Samples arrive in hypothermosol solution. The gonad is washed with PBS and dissociated into single-cells following the protocol below.
?. Fragment the gonads with a scalpel into 2 to 20 pieces per gonad (depending on developmental stage).
?. Suspend gonadal fragments into a 15 mL tube containing of Trypsin-EDTA (0.25%) + DNaseI mix. Volume is depending on tissue size.For small tissues:  in an eppendorff
4 mL
750 µl
?. Place the tube/s into the incubator at for . Rotate and shake to make sure that the gonadal fragments are uniformly distributed on the wall of the tube. For small tissues: incubate epp at  : 5 min incubation + up & down pipetting + 5 min incubation + up & down pipetting +  5 min incubation
37 °C
37 °C
?. If there are still fragments of tissue in suspension, incubate for 5-10 more minutes. Otherwise continue to next step. This will avoid unnecessary damage on the already dissociated cells. Take a note if this is the case.
?. Add  of complete medium with pen-step. Pipette up and down.For small tissues: add up & down pipetting
9 mL
500 µl
?. Filter suspension with a 100 uM filter in 50 ml tube. Top it up with complete medium up 50 ml. For small tissues: Filter suspension in a FACs tub strainer and then transfer it to an eppendorf
?. Centrifuge at 500xg for at
4 °C
?. Discard the supernatant and suspend the cellular pellet in of PBS. Transfer to 1.5 ml eppendorf tube.
1 mL
?. OPTIONAL *If pellet is reddish, add RBC lysis buffer and incubate for 10 min at room temperature.
. *RBC lysis buffer preparation: Dilute 10X RBC lysis buffer stock with water. 
After RBC lysis, add 1 ml of PBS and centrifuge 500 g for 5 min. 
Wash once with PBS 0.04% BSA.
?. Centrifuge at 500xg for at
4 °C
?. Discard supernatant and re-suspend in 250 ul PBS 0.04% BSA. Adjust volume accordingly depending on cell count. For small tissues: re-suspend in 100 ul PBS 0.04% BSA
?. Prepare digestion mix  ABC1ReagentsVolumeConcentration (stock)2Trypsin-EDTA (0.25%) phenol red4ml (depending on tissue size)0.25% (1X)3DNaseI (0.1mg/ml)40ul10mg/ml       Prepare complete media:   ABC1ReagentsFinal conc.2RPMI445ml3FBS50ml10%4Pen-strep5ml1%Prepare final resuspension buffer:  ABC1ReagentsVolumeConcentration (stock)2PBS5ml3BSA 0.04%20ul10%
ABC1ReagentsVolumeConcentration (stock)2Trypsin-EDTA (0.25%) phenol red4ml (depending on tissue size)0.25% (1X)3DNaseI (0.1mg/ml)40ul10mg/ml
ABC1ReagentsFinal conc.2RPMI445ml3FBS50ml10%4Pen-strep5ml1%
ABC1ReagentsVolumeConcentration (stock)2PBS5ml3BSA 0.04%20ul10%
TITLE: Beam Break and Nesting
AUTHORS: Haley   Geertsma

[DESCRIPTION]
This protocol is used to test mice in the Beam Break and Nesting behavioural test.

[STEPS]
2. Single-cage mice in the Beam Break rack with enough food and water for 48-hours.
1. Start Fusion software to begin recording and confirm the lights are on the appropriate light-dark cycle.
3. Turn off Fusion software and add a square of cotton nestlet. Leave mice for 17-19 hours.
4. Record and/or score nests as per https://doi.org/10.1038/nprot.2006.170.
5. Return mice to their home cages.
TITLE: Growing Arabidopsis on nutrient-rich media
AUTHORS: Diep R Ganguly

[DESCRIPTION]
Notes on growing Arabidopsis seedlings on nutrient-rich media in plates.

[STEPS]
SECTION: Prepare media
1. Select and prepare autoclaved media of choice (see recipes below). Make sure to pH before adding gelling agent. 

  MS basal salts	 2.15 – 4.33 g/LSucrose10-30 g/LVitamins (1000X) (optional)1 mL/L	MES (optional)0.1% (w/v)KOHpH 5.75Agar/Phytoblend8 g/L

  Gamborg’s B-5 basal medium3.21 g/LSucrose20-30 g/LKOHpH 5.75Agar8 g/L

  Kanamycin1.65 g/LBASTA (10 g/L	KOHpH 5.75Agar7 g/L

SECTION: Prepare media
2. Pour room temperature media into plates in a biosafety cabinet - keep everything sterile! Allow media to cool and solidify, then package and store at 4 °C. 
SECTION: Surface sterilize seed
3. Collect small aliquots of desired Arabidopsis seeds into clean microfuge tubes.
SECTION: Surface sterilize seed
4. Add 500 uL of a solution made of 40% (v/v) household bleach, and 0.01% (v/v) Tween20 dissolved in distilled water and incubate for 5 minutes.
SECTION: Surface sterilize seed
5. Remove supernatant, then rinse seeds with 700 uL of 70% ethanol incubating for 5-10 minutes. If you did not perform bleach sterilization step, repeat ethanol wash an additional 1-2 times.
SECTION: Surface sterilize seed
6. Remove supernatant then rinse with distilled water at least 3 times.
SECTION: Surface sterilize seed
7. After final rinse, plate seeds out on media plates in sterile environment (e.g. BioSafety cabinet). Seeds can be stored at 4 °C for up to 1 month (maximum).