eamag/protocols-with-steps huggingface dataset:
TITLE: U Mass - Surgery – tail vein injection
AUTHORS: Jason Kim

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary: </span></div><div class = "text-block">Intravenous administration via tail vein is used to acutely deliver drug, hormones, and adenoassociated virus in mice. A large fraction of injectate will be cleared by liver.</div></div>

[STEPS]
?. Place mice in clean cages and keep warm with a heat lamp for ~5 min with constant monitoring to induce vasodilation.
?. Transfer mice to a restrainer with a hole for the tail.
?. Apply 70% Ethanol wipes to tail and turn 45º for a visible presentation of tail vein.
?. Introduce a 27G syringe into tail vein to administer an injectate into mice. (For precise volume introduction, a U-100 insulin syringe can be used to avoid dead volumes.)
?. Apply light pressure over the injection site for ~30 sec to prevent backflow.
?. Return mice to housing cages and monitor for the next several days for any evidence of swelling or complications at the injection site.
TITLE: CTAB-based  DNA extraction for citrus
AUTHORS: Shingo Goto

[DESCRIPTION]
CTAB-based protocols are used for genomic DNA extraction from many kinds of plant species. However, the protocols can’t necessarily completely remove contamination of polysaccharide and RNA in extracted genomic DNA solution. Especially, citrus leaves generally contain high polysaccharide. This protocol is a simple and efficient method for extracting genomic DNA for citrus without contamination of polysaccharide and RNA.

[STEPS]
SECTION: Buffer preparation
1. 2×CTAB solution: 2% (w/v) CTAB, 100mMTris-HCl pH8.0, 1.4M NaCl, 20mM EDTA pH8.0 [1]
High-salt precipitation solution: 1.2M NaCl , 0.8M Sodium citrate [2]
10 mg/ml RNase (Nippon gene)
Chloroform
Isopropanol
70% Ethanol
TE buffer: 10 mM Tris-HCl, 1 mM EDTA, pH 8.0
Reference
Allen GC, Flores-Vergara MA, Krasynanski S et al. A modified protocol for rapid DNA isolation from plant tissues using cetyltrimethylammonium bromide. Nat Protoc 2006;1:2320–5.
Chomczynski P, Mackey K. Modification of the TRI Reagent procedure for isolation of RNA from polysaccharide- and proteoglycan-rich sources. Biotechniques 1995;19:492–5.
SECTION: Homogenization and cell lysis
2. Preheat 2×CTAB solution to 60 °C  in water bath. Add  2 % (v/v)  of 2-Mercaptoethanol to the 2×CTAB solution just before use.

SECTION: Homogenization and cell lysis
3. Homogenize 100 mg of fresh leaf in liquid Nitrogen. Add 800 µL of 2×CTAB solution and completely suspend homogenized leaf. Transfer the suspended solution to 2 ml tube.

SECTION: Homogenization and cell lysis
4. Add4 µL  of 10 mg/mL  RNase, mix by inversion, and incubate at 37 °C  for 15 min  (In order to prevent RNase contamination in laboratory, RNase treatment is conducted before denaturing proteins by chloroform).

SECTION: Homogenization and cell lysis
5. Incubate at 56 °C for 30 min  inverting the tube once every 10 min.

SECTION: Chloroform extraction
6. Add 300 µL of Chloroform and mix gently with tube rotator for 15 min.

SECTION: Chloroform extraction
7. 13000 rpm, 10 min, 25 °C 
SECTION: Chloroform extraction
8. Transfer supernatant carefully to new 2 ml tube.
SECTION: Chloroform extraction
9. Repeat step 6 and 7
SECTION: Precipitation and wash of DNA pellet
10. Transfer 600 µL of supernatant to new 1.5 ml tube, add 300 µL of High-salt precipitation solution, and mix by inversion.

SECTION: Precipitation and wash of DNA pellet
11. Add 300 µL of Isopropanol and mix by inversion.

SECTION: Precipitation and wash of DNA pellet
12. 15000 rpm, 10 min, 25 °C 

SECTION: Precipitation and wash of DNA pellet
13. Discard and remove supernatant (DNA pellet is often transparent).
SECTION: Precipitation and wash of DNA pellet
14. Add 1000 µL of 70% ethanol and mix by inversion 10 times to wash salts.

SECTION: Precipitation and wash of DNA pellet
15. 15000 rpm, 5 min, 25 °C 

SECTION: Precipitation and wash of DNA pellet
16. Completely remove supernatant and dry up DNA pellet in air.
SECTION: Precipitation and wash of DNA pellet
17. Dissolve pallet in 100 µL of TE buffer.

SECTION: Result of Gel electrophoresis
21.  
  

SECTION: Result of gel electrophoresis
18.
TITLE: Do the proportion of protocol registrations about systematic reviews outside PROSPERO in medical and biomedical sciences increase over the years?: a meta-epidemiological study protocol
AUTHORS: Masahiro Banno, Yasushi Tsujimoto, Yuki Kataoka

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Objectives. To evaluate how often researchers registered protocols about systematic reviews (SRs) outside PROSPERO in medical and biomedical sciences. </div><div class = "text-block">Methods. This study will be a meta-epidemiological study. We will include all the protocols about SRs in PROSPERO, 44 preprint severs, Protocols.io, ClinicalTrials.gov and International Clinical Trials Registry Platform (ICTRP), OSF Registries since 2011. We will retrieve titles and its publication years and whether the theme of preprints is related with COVID-19. We will describe the proportion of SRs protocol registration in the platforms outside PROSPERO over the years. We will display the trends. We will also compare characteristics of random samples of SRs protocols registered on PROSPERO with those registered outside PROSPERO.</div><div class = "text-block">Ethics & Dissemination. This study does not need ethics approval. We registered this study protocol. We will publish the findings in a peer-reviewed journal and may present them at conferences. </div><div class = "text-block">Discussion. This study might clarify whether proportion of protocol registrations about systematic reviews to platforms outside PROSPERO in medical and biomedical sciences increase over the years and whether systematic reviewers need to search these data sources before conducting SRs.</div><div class = "text-block">Registration: Protocols.io</div></div>

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Luminex Mulliplex Cytokine/Chemokine 17-plex MAG manufacturer's protocol</p>

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>A description of to how to produce and evaluate three-dimensional computerized forensic craniofacial reconstructions (CCFR) using Horos, Blender, Cloud Compare, MakeHuman and Picassa computer programs.</p>

[BEFORE_START]
<p>You need download the software programs: Horos, Blender, MakeHuman, CloudCompare and Picasa.</p>
<p>You need a computed tomography (CT) of the skull of the alive person.</p>

[STEPS]
?. 
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TITLE: Study Design (Part 3 of Phase 3 study of Vaccine Candidate for COVID-19)
AUTHORS: Chris Ockenhouse, Chris Gast, Renee Holt, Jorge Flores

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This is Part 3 of "Phase 3 randomized, double-blinded, placebo-controlled trial to evaluate the safety, immunogenicity, and efficacy of </span><span style = "font-weight:bold;">Vaccine Candidate</span><span> against COVID-19 in adults > 18 years of age"</span></div><div class = "text-block">This generic Phase 3 protocol was developed by the PATH team with support of the Bill and Melinda Gates Foundation. The aim of the collection is to share recommended best practices in designing and implementing a Phase 3 study of a COVID-19 vaccine candidate. As Phase 3 trials of different Vaccine Candidates  proceed around the world, following the same protocols will ensure consistency and comparability of the Phase 3 trial results.</div><div class = "text-block"><span style = "font-weight:bold;">Please note</span><span> that this is an evolving document, to be versioned and updated, based on community feedback and new data.</span></div></div>

[STEPS]
TITLE: DigiGait
AUTHORS: Haley   Geertsma

[DESCRIPTION]
This protocol is used to test mice in the DigiGait behavioural test.

[STEPS]
1. Habituate mice in the testing room for 60 minutes.
2. Place mice on the DigiGait treadmill and start the treadmill at 18-22cm/s. Record their movement for 3 seconds then place them back into their home cage.
3. Analyze 3-second clips with DigiGait Analysis Software.
TITLE: Synapto-iATPSnFR2-miRFP670nano3 analysis
AUTHORS: Alexandros C Kokotos, Tim Ryan

[DESCRIPTION]
This protocol describes how to analyze imaging experiments using Synapto-iATPSnFR2-miRFP670nano3 from primary neurons.

[STEPS]
SECTION: Synapto-iATPSnFR2-miRFP670nano3 image analysis
1. Open the image stacks to by analyzed with ImageJ by drag and drop.
SECTION: Synapto-iATPSnFR2-miRFP670nano3 image analysis
2. Using the far-red miRFP670nano3 channel and blind to the ATPSnFR2 channel, identify nerve terminals by their appearance as fluorescent puncta.
SECTION: Synapto-iATPSnFR2-miRFP670nano3 image analysis
3. Place ROIs on the nerve terminals using the Time Series Analyzer plug-in. 
We usually use ROIs of width and height 6 pixels each, with the pixel size being 400 nm in our images.
SECTION: Synapto-iATPSnFR2-miRFP670nano3 image analysis
4. Get the average pixel intensity of each ROI and each image using the "Get Average" function. Save the produced csv results as well as the ROIs selection.
SECTION: Synapto-iATPSnFR2-miRFP670nano3 image analysis
5. Similarly place background ROIs surrounding the nerve terminals identified. Save the pixel intensity values and background ROIs selection.
SECTION: Synapto-iATPSnFR2-miRFP670nano3 data analysis
6. Open the produced csv files in Microsoft Excel.
SECTION: Synapto-iATPSnFR2-miRFP670nano3 data analysis
7. For each time point, subtract the average background fluorescence values from every single nerve terminal fluorescence values.
SECTION: Synapto-iATPSnFR2-miRFP670nano3 data analysis
8. For each time point, calculate the ratio of the iATPSnFR2 signal to the miRFP670nano3 signal, for every nerve terminal. This will yield the Fratio for all single nerve terminals.
Note: Since the miRFP670nano3 signal remains stable throughout the experiment, the iATPSnFR signal can be also normalized to the initial miRFP670nano3 signal before action potential train, to minimize the noise of the produced traces.
SECTION: Synapto-iATPSnFR2-miRFP670nano3 data analysis
9. Calculate the average Fratio of all the nerve terminals of one single neuron.
SECTION: Synapto-iATPSnFR2-miRFP670nano3 data analysis
10. The initial Fratio can be used as a reading of the baseline ATP of single cells before the action potential trains.
SECTION: Synapto-iATPSnFR2-miRFP670nano3 data analysis
11. The data-set can be further normalized to 1, by dividing Fratio to the initial Fratio values, for each individual cell. This normalization allows to specifically observe ATP kinetics during and after action potential trains and is unaffected by the individual cell baseline ATP.
SECTION: Synapto-iATPSnFR2-miRFP670nano3 data analysis
12. The normalized Fratio at the end of the action potential train as well as the recovery of ATP at a later time point can be calculated for all single cells.
SECTION: Synapto-iATPSnFR2-miRFP670nano3 data analysis
13. All produced time trace data and subsequent quantifications can be plotted and presented in Graphpad Prism.
TITLE: Female Reproductive System Deceased Donor and Tissue Eligibility Criteria for Penn TMC
AUTHORS: kone  

[DESCRIPTION]
This document outlines the required criteria for deceased donor inclusion of the female reproductive system in the Human BioMolecular Atlas Program (HuBMAP) for the University of Pennsylvania Tissue Mapping Center.This protocol was developed and is executed in collaboration with the organ procurement organization the Gift of Life Donor Program.

This study intends to be as inclusive as possible of race and ethnicity.

[STEPS]
1.  Gift of Life Donor Program (GLDP) transplant coordinators receive annual training by MPI O’Neill in HuBMAP initiative.
2. Trained coordinators will evaluate patient for eligibility using criteria detailed in following sections.
3. Transplant coordinator will call MPI O’Neill to review inclusion/exclusion criteria and confirm sample team availability.
4. Once MPI O’Neill has confirmed donor meets I/E criteria and sample team is available, LNOK (living next of kin) will be approached by transplant coordinators to obtain authorization for vascularized composite allograft (VCA) donation and use of tissue for research purposes. See  .

5. Provided LNOK consents, procurement team will be notified of donor enrollment in HuBMAP and the female reproductive system will be recovered.
SECTION: Inclusion criteria
6. Authorization for organ and/or tissue donation is signed by living next of kin (LNOK). See  .
Donor has known state of health and diagnoses
Donor has non-reactive HIV/HCV/HBV NAT testing documented.
Donor has non-reactive HBV surface antigen testing documented.
Donor has non-reactive HBC encoded antigen testing documented.
Organ donation is after brain death
Donor has normal gross Mullerian structures
Donor is between the 18-45 years of age

SECTION: Exclusion criteria
7. Organ donation after cardiac death
Pregnancy
History of reproductive tract malignancy, endometriosis, significant uterine surgery (specifically myomectomy)
Tissue collected >4 hours after clamp time.
TITLE:           ELISA for quantification of human C9 in serum or plasma.
AUTHORS: Angel  Justiz-Vaillant, Belkis Ferrer-Cosme

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

[STEPS]
?. An anti-human C9 coating antibody is adsorbed onto the microwells by incubation overnight at 4°C with carbonate-bicarbonate buffer.
?. Add 50 µl of human serum or plasma. Human C9 present in the serum or plasma binds to antibodies adsorbed into the microwells.
?. The microplate is blocked with 3% non-fat milk-PBS buffer and later wash to remove unbound proteins.
?. Fifty (50) µl of biotin-conjugated anti-C9 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-C9 antibody. The optimal dilution of this conjugate must be investigated.
?. The plate is washed following incubation to remove the unbound Streptavidin-HRP.
?. Add 100 µl of 3,3',5,5'- tetramethylbenzidine (TMB; Sigma-Aldrich) into each well.
?. Incubate the microwells in the dark for 15 min.
?. A colored product is formed in proportion to the quantity of C9 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 C9 standard dilutions and the human C9 sample concentration is determined.
TITLE: TS Spurrs - primary fixation Karnovsky&#39;s - tissue (TM - 013)
AUTHORS: sandra.crameri

[DESCRIPTION]
This method is used for conventional processing of tissue to Spurr's resin.

[GUIDELINES]
All time are minimum times, it is acceptable to go over time specified for any given step. Good place steps to leave overnight or at 70% ethanol and 50/50 Spurrs/Ethanol mix.

[STEPS]
SECTION: HEADER
1. SAN:


SPEC No:


OPERATOR & STEPS:


OPERATOR & STEPS:
SECTION: CONVENTIONAL
2. 2.5 % volume   plus 4 % volume    in 0.1 Molarity (m) for at least 40 min 

SECTION: CONVENTIONAL
3. Wash 0.1 Molarity (M) Sorenson's Phosphate Buffer pH 07.2 (300mosmol/kg) for 15 min 

SECTION: CONVENTIONAL
4. 1 % volume   in buffer for 60 min 

SECTION: CONVENTIONAL
5. 70 % volume   for at least 20 min 

SECTION: CONVENTIONAL
6. 95 % volume   for 20 min 

SECTION: CONVENTIONAL
7. 100 % volume   for 20 min 

SECTION: CONVENTIONAL
8. 100 % volume   dried molecular sieved for 20 min 

SECTION: CONVENTIONAL
9. 50 % volume   50 % volume   for 30 min 


SECTION: CONVENTIONAL
10. 100 % volume   30 min 

SECTION: CONVENTIONAL
11. 100 % volume   60 min 

SECTION: CONVENTIONAL
12. Oven polymerise overnight at 65 °C
TITLE: protein extraction from cell pallets
AUTHORS: Jacob   Waldbauer, Lichun Zhang

[DESCRIPTION]
Use LDS buffer to extract protein from cell palllets

[STEPS]
1. Make 1X LDS buffer. In 40mL of 1X buffer, add Tris HCl 0.666g, Tris base 0.682g, LDS 0.8g, EDTA 0.006g, Glycerol 4g.
2. make 1M DTT stock solution in LC/MS water.
3. Add 200~500uL of 10mM DTT in 1X LDS buffer to the cell pallet tube. 
4. Heat sample up at 95ºC for 20 minutes.
5. Cool down the sample to room temrature. Meanwhile, make a 0.5M iodoacetamide stock solution.
6. Add IAM to 50mM/60mM in the sample, incubate in dark for 30-60minutes.
7. Add DTT to 50mM/60mM. Sampe is ready to continue with FASP.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol describes how to isolate bacteria in order to label its biomass using <sup>13</sup>C-glucose and <sup>15</sup>N-ammonia. The first steps aim to isolate candidate bacteria that are able of growing on the chosen substrates for isotopic labeling.</p>
<p> </p>
<p>The protocol was applied to the isolation of bacetria from freshwater algal cultures, although it can be applied to natural samples.</p>
<p> </p>
<p>The M9 media used in the protocol is based on the media from Marley, Lu and Bracken (2001).</p>
<p> </p>
<p>References:</p>
<p> </p>
<p>Marley, J., Lu, M., &amp; Bracken, C. (2001). A method for efficient isotopic labeling of recombinant proteins. <em>Journal of Biomolecular Nmr</em>, <em>20</em>(1), 71–75.</p>

[STEPS]
?. 
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TITLE: RPA DNA Amplification using TwistAmpTM Basic
AUTHORS: Luc Gabel, Théo Nass

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol describes the procedure to perform an RPA DNA Amplification using the TwistAmp</span><span style = "vertical-align:super;">TM</span><span> Basic</span></div></div>

[STEPS]
?. Prepare the RPA pellet rehydration solution in a 1.5 ml tube following the table in the Materials section.If possible do a Master Mix that will then be divided.
?. For each sample, transfer the entire volume (47.5 µl) to a reaction pellet. Pipette up and down until the pellet is fully resuspended.
?. Add 2.5 µl of 280mM MgOAc to the cap of each reaction tube. MgOAc will make the reaction start, it is added to the cap so that all reactions start simultaneously when spun down.
?. Incubate at 39°C for 20 minFor low copy numbers, incubate 4 minutes, vortex and spin briefly and incubate again for 16 minutes.
?. After the incubation, purify the sample if you want to run the RPA product on an agarose gel.PCR purification kits work well, but since RPA primers can be very long (60+ bp), a high cutoff is recommended.Do not open post-amplification RPA reactions in the same workspace as you set up pre -amplification RPA, or take appropriate measures to limit DNA contamination.
?. Do a short spin to make the MgOAc drop fall down into the tube, vortex briefly and do another quick spin.
?. Vortex and spin briefly.
TITLE: IB BIO SL IA M23 - A study on the conditions that affect yeast fermentation 
AUTHORS: Marcy  Roca Cugat

[DESCRIPTION]
This protocol is designed to aid me in the research for my International Baccalaureate Biology Internal Assignment

[GUIDELINES]
None

[STEPS]
SECTION: Preparation
1. Check that all reagents and materials are ready.
SECTION: Preparation
2. Assemble the instrumentation
SECTION: Preparation
2.1. One end of the flask must be plugged with a holed stopper and a thermometer.
The other end should be plugged with a holed stopper and a glass tube.
The middle opening should be covered with plastic and secured with a rubber band.
SECTION: Preparation
2.2. A long and shallow recipient must be filled with water as much as possible. The gas recovery flask should also be filled with water as much as possible. Then, the latter should be inverted and suspended inside the former.
SECTION: Preparation
2.3. The tube should be connected from the glass tube to the gas recovery flask. 
SECTION: Preparation
3. The block of compressed yeast must be cut in 4 parts
SECTION: Processing
4. Several factors must be checked and accounted for: pressure, temperature and initial weight of the full gas recovery flask.

SECTION: Processing
5. One quarter of the yeast block must be dissolved in sugary water (concentration undetermined, volume undetermined, temperature undetermined). This should then be placed in the flask.

SECTION: Processing
6. Wait for fermentation: 30 min 

SECTION: Processing
7. Weigh the gas recovery flask. The difference from the first measurement is the gas generated. Convert it to moles of CO2+H2O for a universal measurment
TITLE: Artificial Cerebrospinal Fluid VII (ACSF.VII)
AUTHORS: Allen Institute for Brain Science

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is used to prepare Artificial Cerebrospinal Fluid VII (ACSF.VII). ACSF.VII is used for applications including fresh human brain tissue transportation and as a bathing solution during fresh human brain slicing.</div><div class = "text-block"><span style = "font-weight:bold;">Note</span><span>: Research reported in this publication was supported by the National Institute Of Mental Health of the National Institutes of Health under Award Number U01MH114812. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.</span></div></div>

[STEPS]
TITLE: Efficient recovery of complete gut viral genomes by combined short- and long-read sequencing
AUTHORS: chuqingsun

[DESCRIPTION]
Current metagenome-assembled human gut phage catalogs contained mostly fragmented genomes. Here, we developed a vigorous gut virome detection procedure involving viral-like particle enrichment from increased amount of feces (~500g) and combined sequencing of short- and long-reads. Applied to 135 fecal samples, we assembled a Chinese Gut Virome Catalog (CHGV) consisting of 21,646 non-redundant phage genomes that were significantly longer than those obtained by short-read sequencing and contained ~35% complete ones, which was ~nine times more than those in the Gut Virome Database (~4%). Interestingly, majority (~60%) of the CHGV genomes were obtained by either long-read or hybrid assemblies, which overlapped little with those assembled from only the short-reads, indicating the necessity of combined sequencing in gut virome discovery. With this dataset, we elucidated the vast diversity of the gut virome from several aspects, including the identification of 32% novel genomes as compared with public gut virome databases, dozens of phages that were more prevalent than the crAssphages and/or Gubaphages, and several viral clades that are more diverse than the two. In the end, we also characterized the functional capacities of the CHGV encoded proteins and constructed a viral-host interaction network to facilitate future research and applications of the gut viruses.

[STEPS]
SECTION: Assembly
1. NGS Assembly
 

SECTION: Assembly
2. # TGS assembly
 


SECTION: Assembly
3. Hybrid Assembly

 

SECTION: Additional analysis
4. Viral annotation

 

SECTION: Additional analysis
5. UHGG filteration

 


SECTION: Additional analysis
6. checkv

 

SECTION: Additional analysis
7. RPKM calculation
TITLE: Collection of GBS samples at 35-37 weeks of gestation and isolation
AUTHORS: lydiariver  

[DESCRIPTION]
Recto- vaginal samples collections usually practice to pregnant women because Streptococcus agalactiae  a bacterial pathogen frequently carried in the normal fecal and/or vaginal flora but also is one of the most importants pathogens involve on neonates infections. 

[STEPS]
1. Remove swab from packaging and insert swab 2 cm into vagina, followed by the rectum, insert swab 1 cm through the anal sphincter using the same swab. 
 

2. Place the swab (s) into a nonnutritive transport medium. Apropriate transport systems (e.g, Stuarts or Amies with or without charcoal) are commercialy available. 
 

3. Specimen requisitions should indicate clearly that specimes are for group B Streptococcal testing. 
4. For sample processing, the swab is removed from the transport system and inoculated into blood agar plates, streaked to isolate, and incubated at 37 °C for 1440 min