eamag/protocols-with-steps huggingface dataset:
TITLE: ChIP-seq
AUTHORS: José Terrón-Bautista, Francisco Gómez-Vela, Irene Delgado-Sainz, Federico Divina, Miguel Garcíıa-Torres, Pedro Manuel Martínez-García

[STEPS]
?. [Crosslinking]
Fix cells adding formaldehyde to a final concentration of 1% and incubation at 37ºC for 10 min.
?. [Crosslinking]
Quench fixation adding glycine to a final concentration of 125 mM at cell culture plates and incubate at RT for 5 min.
?. [Crosslinking]
After two washes with cold PBS, in the presence of complete protease inhibitor cocktail (Roche) and PMSF, scrape cells and centrifuge at 4ºC for 5 min at 300 g.
?. [Cell lysis and chromatin sonication]
Add 2,5 ml of 0.5% NP-40 lysis buffer to lyse cell pellet for nuclei isolation and incubate 10 min.
?. [Cell lysis and chromatin sonication]
Centrifuge 5 min 3000g 4ºC
?. [Cell lysis and chromatin sonication]
Resuspend cell pellet in 1 ml SDS 1% lysis buffer.
?. [Cell lysis and chromatin sonication]
Sonicate cells in lysis buffer using Bioruptor (Diagenode, UCD-200) at high intensity for three cycles of 10 min with 30s sonication and 30s pause.
?. [Cell lysis and chromatin sonication]
Clarify chromatin by centrifugation at 17000g for 10 min at 4ºC
?. [IP]
Incubate 30 ug of chromatin with 4 ug of antibody overnight at 4ºC in IP buffer (0.1% SDS, 1% 161 TX-100, 2 mM EDTA, 20 mM TrisHCl pH8, 150 mM NaCl) with rotation.
?. [IP]
Add 25 ul of pre-blocked (1 mg/ml BSA) Dynabeads protein A and Dynabeads protein G (ThermoFisher) for 4 hours.
?. [IP]
Wash beads with IP buffer for 10 min.
?. [IP]
Wash beads with IP buffer with increased salt concentration (500 mM NaCl) for 10 min.
?. [IP]
Wash beads with LiCl buffer (0.25 M LiCL, 1% NP40, 1% NaDoc, 20 165 mM TrisHCl pH8 and 1 mM EDTA) for 10 min.
?. [IP]
ChIPmentation was carried out as described in Schmidl, 2015 using Tagment DNA Enzyme provided by the Proteomic  Service of CABD (Centro Andaluz de Biología del Desarrollo).
?. [IP]
Elute DNA from beads by incubation at 50ºC in 100 ul elution buffer (1% SDS and 100 mM NaHCO3) for 30 min.
?. [De-crosslinking and purification]
Incubate eluted samples with 200 mM NaCl and 100 ug/ml of Proteinase K (ThermoFisher) overnight at 65ºC to revert crosslinking.
?. [De-crosslinking and purification]
Purify DNA using Qiagen PCR Purification columns.
?. [Library Preparation]
Amplify DNA for N-1 cycles (being N the optimum Cq determined by qPCR reaction) using standard protocol of NEBNext High-Fidelity Polymerase (M0541, New England Biolabs) in order to generate libraries.
?. [Library Preparation]
Purify libraries and size-select using Sera-Mag Select (GE Healthcare). DNA not bound to 0,7x volumen of Sera-Mag beads were subsequentially incubated with 0,15x volumen of Sera-Mag beads. In this way, DNA fragments between 100-500 bp were isolated. Libraries were sequenced using Illumina NextSeq 500 in a single-end configuration.
TITLE: Glycemic effect of post-meal walking compared to one prandial insulin injection in type 2 diabetic patients treated with basal insulin: a randomized controlled cross-over study
AUTHORS: Onnicha Suntornlohanakul, Chatchalit Rattarasarn, Atiporn Ingsathit, Chatvara Areevut, Sunee Saetung

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Studies demonstrate that post-meal walking decreases postprandial hyperglycemia in type 2 diabetic patients but it has never been tested with the active treatment comparator. The objective of this study was to determine the effect of post-meal walking on glycemic control compared with one prandial insulin in type 2 diabetic patients who failed basal insulin. A randomized controlled cross-over study of post-meal walking or one prandial insulin was done in type 2 diabetic patients who were being treated with basal insulin between May 2017 and March 2018. In post-meal walking group, patients walked after meal for 15-20 minutes one meal a day every day for 6 weeks. In prandial insulin (basal plus) group, one prandial insulin was injected before breakfast or main meal with rapid-acting insulin. The primary outcome was a difference in HbA1c reduction in post-meal walking compared with basal plus groups. Fourteen patients completed the study. By intention-to-treat analysis, HbA1c was reduced by -0.05(range:-1.08 to 0.74) and -0.19(range:-0.8 to 0.56) % in post-meal walking and basal plus groups respectively. By per-protocol analysis, post-meal walking and basal plus groups decreased HbA1c by 0.13(range:-0.74 to 1.08) and 0.2(range:-0.56 to 0.8) %, respectively. There was no significant differences in HbA1c reduction from baseline in each group and between groups in both intention-to-treat and per-protocol analysis. Fructosamine levels were decreased by 17.5(-59 to 43) and 10(-15 to 40) µmol/L, respectively at 3 and 6 weeks in post-meal walking group whereas the respective changes in basal plus group were 12.5(-17 to 64) and 17.5(-28 to 38) µmol/L and there was no significant difference between groups. In conclusion, although post-meal walking might be as effective as one prandial insulin to improve glycemic control in type 2 diabetic patients who failed basal insulin but the magnitude of reduction was small. A longer-term study with a larger sample size or with a different walking protocol is required.</div></div>

[STEPS]
TITLE: Protocol  PCR Wet Lab 
AUTHORS:  

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

[STEPS]
?. DNA ExtractionDNA Extraction: Saliva (Bento lab)  This protocol extracts DNA from Saliva using a microcentrifuge and thermocycler. The microcentrifuge separates the cells in the sample into a pellet at the bottom of the microcentrifuge tube. Extra liquid is drawn out of the microcentrifuge tube. Cells are then heated in the thermocycler and placed in the microcentrifuge to separate DNA from other cellular material.
?. PCRLactose intolerance: PCR (Bento lab)Bitterness tasting: PCR (Bento lab)Athlete gene: PCR (Bento lab)This protocol uses a thermocycler in order to amplify sections of DNA genes. Students will amplify genes specific to lactose intolerance, bitterness tasting, and an athletic gene. DNA template is extracted from the previous protocol and added to a PCR master mix and designated primer. PCR master mix is composed of Taq polymerase, dNTPs, MgCl2, and pH buffer. After adding all reagents, students will set parameters for the thermocyclers. Each PCR experiment will have different parameters due to the nature of its respective primers.
?. Gel ElectrophoresisGel electrophoresis (Bento lab)This protocol uses a gel electrophoresis to identify the amplified sequences from the previous protocol, PCR. Students will set up their gel electrophoresis by creating an agarose gel with necessary lanes, adding 0.5X TBE buffer, and attaching electrodes. After set up, DNA ladders and amplified DNA will be added to lanes in the gel. The gel will run at 50V for 40 minutes. Then students will analyze the results and determine their allele type and phenotype.
?. Take a picture of your gel electrophoresis results and upload it to your Lab Notebook.
TITLE: Set Shifting ASAP Operant Behavior_LernerLab
AUTHORS: jillian.seiler

[DESCRIPTION]
Protocol for set shifting task used by the Lerner Lab for ASAP work

[STEPS]
SECTION: Magazine Training
1. Food restrict animals overnight prior to beginning magazine training. For the remainder of training animals should be restricted to 85% of their free feeding weight.
SECTION: Magazine Training
3. Place animals in operant box with dummy patch cords attached and allow ~5 minutes to habituate
SECTION: Magazine Training
4. Load JN_ASAP_A_MagTraining_30min into MED-PC and start program. The script for the program is attached as a comment. The overview is as follows:

SECTION: Magazine Training
2. Connect 20 mL syringe with >10 mLs of 1:3 diluted sweetened condensed milk. Add ~15 inches of tubing with tubing adapter to syringe. Load in pump, attach to reward port. Prime pump. 
SECTION: Magazine Training
4.1. House light, fan, corner lights, and cue lights above the left and right nosepoke ports are turned on.
SECTION: Magazine Training
4.2. On average, every 60 seconds a noncontigent reward is delivered
SECTION: Magazine Training
4.3. The animal must consume the reward (or make a port entry) in order for the next reward to be delivered
SECTION: Magazine Training
4.4. After 30 minutes, the program ends
SECTION: Magazine Training
5. This program occurs once, regardless of performance
SECTION: Magazine Training
6. After animal has finished, record weight, return to homecage and clean operant chamber tray with 70% ethanol
SECTION: Magazine Training
7. Flush reward port and tubing with ethanol and hang to dry
SECTION: Magazine Training
8. Store any remaining sweetened condensed milk in refrigerator 
SECTION: FR1 Both
9. Repeat steps 2+3   

SECTION: FR1 Both
10. Load JN_ASAP_B_FR1_BOTH and start program. The script for the program is attached as a comment. The overview is as follows:
SECTION: FR1 Both
10.1. Fan and Near-Infrared Lights are turned on
TITLE: Zooarchaeology by Mass Spectrometry (ZooMS)- Pretreatment protocols for bone material
AUTHORS: Samantha Brown, Sandra Hebestreit, Naihui Wang, Nicole Boivin, Katerina Douka, Kristine Richter

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This collection details some of the different established protocols for Zooarchaeology by Mass Spectrometry (ZooMS) for use on archaeological bone.  ZooMS allows for taxonomic identification by the peptide mass fingerprinting of collagen type I.  These protocols can be used individually or combined depending on the preservation, sample size, and ability to do destructive analysis.   All protocols are optimized for bone as the starting material.</div><div class = "text-block">The AmBic protocol can be used on samples where distructive analysis cannot be undertaken.  Samples are pretreated by soaking in ammonium bicarbonate at room temperature followed by a brief heating step to "melt" a small amount of collagen out of the bone.  The bone can then be dried at room temperature.</div><div class = "text-block">Both acid based protocols are destructive as the samples are pretreated with hydrocholric acid to demineralize the bone.  In the acid soluble protocol the acid is removed and the collagen is filtered from the acid.  In the acid insoluble protocol the bone shaddow that remains after demineralization is washed to remove the acid and then heated in ammonium bicarbonate to gelitinize the collagen.  Both acid based protocols can be done on the same sample in conjunction with each other and then either analyzed separately on the MALDI or combined before analysis.</div><div class = "text-block">In all protocols the extracted collagen is then digested with trypsin and the peptides are purified using C18 ZipTips.</div><div class = "text-block">If you are using any of the protocols please cite the DOI for the protocol, the following paper, and any papers in the additional individual protocols: </div><div class = "text-block"><span>Buckley, M., Collins, M., Thomas-Oates, J., & Wilson, J. C. (2009). Species identification by analysis of bone collagen using matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry. </span><span style = "font-style:italic;">Rapid Communications in Mass Spectrometry: RCM</span><span>, </span><span style = "font-style:italic;">23</span><span>(23), 3843–3854. https://doi.org/10.1002/rcm.4316</span></div></div>

[STEPS]
TITLE: UC Davis - Microvascular Permeability and Lipoprotien Flux
AUTHORS: John Rutledge

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary: </span></div><div class = "text-block">One of the three indices of arterial function that are compromised to a varying degree in individuals with cardiovascular disease is vascular permeability. This assay measures vascular permeability (as flux of labeled large molecular weight molecules: i.e. albumin or dextran) and lipid permeability (as flux of labeled lipid) in coronary or carotid arteries.</div></div>

[STEPS]
?. Mice are anesthetized with an intraperitoneal injection with 50 mg pentobarbital/kg weight.
?. All treatments are administered into the left femoral vein by bolus injection. FITC-albumin (40 mg/mL) in 100 µL:    a. PBS     b. VLDL (150 mg/dL)     c. VLDL (150mg/dL) + LpL (2 U/mL)     d. LpL (2 U/mL) in PBS
?. Alternatively, the mouse is then infused at with 100 uL fluorescently labeled compound alone (FLC, see above) (40 mg/mL)
?. Excess FLC was removed from the vasculature by infusion with DMEM media for 20 min by infusion into the left ventricle of heart and followed by infusion of 10% formaldehyde for 20 min.
?. The microvascular rich tissues interest are immediately removed and fixed in 10% formaldehyde for two days.         a. microvascular tissues = brain, heart, and mesentery ect.         b. macrovascular tissues= common carotid arteries or aorta
?. The tissue is embedded in paraffin and sectioned to 5 µm thickness.
?. Tissues sections are deparaffinized, rehydrated, and imaged using fluorescent microscopy.
TITLE: Downstream Analysis of ebFRET Data
AUTHORS: Clark Fritsch

[DESCRIPTION]
This protocol follows from the "Using ebFRET for Hidden Markov Modeling" protocol and is the fourth and final step towards analyzing your single-molecule FRET traces using Hidden Markov Modeling. In this protocol, I describe some of the questions that you may ask of the data generated by ebFRET and list some of the programs that I have made to do the calculations required to answer these questions.

[STEPS]
1. After creating your "2stateHMM_practice_Analysis.dat", as described in the "Using ebFRET for Hidden Markov Modeling" protocol, you can then calculate / retrieve various types of information from your data.
2. The downstream analysis that I have done so far for the ebFRET data can be divided into the following questions:

1. What is the distribution of FRET values for each state that was identified by ebFRET?

  

2. What is the idealized FRET efficiency for each FRET state that was identified by ebFRET? 

  

3. What is the actual FRET efficiency (non-idealized FRET) for each FRET state that was identified by ebFRET? 

  

4. What is the lifetime of each FRET state that was identified by ebFRET? Outputs a file that can be used in MEMLET to determine rate constants for each state.

  

3. Each of the programs included above were made for simple ad-hoc analysis of the data outputted from ebFRET and will likely need to be modified slightly depending on the number of states that you had ebFRET fit to your data.
TITLE: LB borth or agar
AUTHORS: Adrien Assie, Buck Samuel

[DESCRIPTION]
LB broth or agar recipe

[STEPS]
1. Start with 900 mL

2. 10 gBacto-tryptone

3. 5 g Bacto-yeast

4. 5 gNaCl

5. Adjust pH to 7.0 using 5 Molarity (M)NaOH

6. For LB Agar add 15 g 

7. Adjust water to 1000 mL

8. Autoclave
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<div class="row bibliography-item-info">
<div class="bibliography-item-copy-text content col-md-12" data-clipboard-target="copy-target-228943496" data-redirect-target="/items/228943496/copy">Adapted from: Van Etten, J. (n.d.). Titering of <em>Chlorella </em>Viruses. Retrieved from http://ncv.unl.edu/vanettenlab/</div>
<div class="bibliography-item-copy-text content col-md-12" data-clipboard-target="copy-target-228943496" data-redirect-target="/items/228943496/copy">Contact Dr. Steven Wilhelm (wilhelm@utk.edu) or Samantha Coy (srose16@vols.utk.edu) for additional information regarding this protocol.</div>
</div>

[STEPS]
?. 
?. 
?. 
?. 
?.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Commonly used protocol to isolate peripheral blood mononuclear cells from whole human blood or apheresis packs</p>

[BEFORE_START]
<ul>
<li>Make sure to repeatedly label sample with donor number, especially if working with multiple donors</li>
<li>The protocol here is optimized for 10ml of material from platelet apheresis collars. Variations for other sources have been described.</li>
</ul>

[GUIDELINES]
<p><span style="font-weight: 400;"><strong>Objective: </strong>Isolate peripheral blood mononuclear cells from fresh whole blood or apheresis packs, also referred to as Leukopaks or collars.In our case, these are platelet-depleted samples of human blood given from a donor. These can vary in volume and cell composition. </span></p>
<p> </p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: Reverse transcription using SuperScript IV
AUTHORS: Amin Mahpour

[STEPS]
?. Mix the following reagents from the kit. Please scale up if more RT reaction is desired.  AB1ReagentAmount (uL)2Primer (Random or dT)0.53dNTP (10mM)14RNA11
AB1ReagentAmount (uL)2Primer (Random or dT)0.53dNTP (10mM)14RNA11
?. Incubate the mixture at   for . Then, incubate samples on ice for few minutes.
72 °C
This step allows denaturation of RNA and proper priming for the downstream cDNA synthesis.
?. Mix the following reagents from the kit. Please scale up if more RT reaction is desired.  AB1ReagentAmount (uL)2RT Buffer (5x)43DTT(10mM)14RNase Inhibitor1Add the 6uL to the 12.5uL mix from Step 3.
AB1ReagentAmount (uL)2RT Buffer (5x)43DTT(10mM)14RNase Inhibitor1
?. Incubate the samples at  for . Then, add 1.5uL SuperScript RT IV enzyme to the reaction and mix well.
37 °C
?. Incubate the samples using the following incubation settings:  AB1Temp (C)Time (minutes)2255345404551057510
AB1Temp (C)Time (minutes)2255345404551057510
?. Add 1uL RNase H to the cDNA samples and incubate at  for .
37 °C
?. Dilute the cDNA samples using Nuclease free water.
TITLE: No Title
AUTHORS: 

[STEPS]
SECTION: Cell Culture
?. 
SECTION: Fixation, permeabilization, and staining
?. 
SECTION: Fixation, permeabilization, and staining
?. 
SECTION: Fixation, permeabilization, and staining
?. 
SECTION: Fixation, permeabilization, and staining
?. 
SECTION: Fixation, permeabilization, and staining
?. 
SECTION: Fixation and permeabilization
?. 
SECTION: Fixation, permeabilization, and staining
?. 
SECTION: Fixation, permeabilization, and staining
?. 
SECTION: Fixation, permeabilization, and staining
?. 
SECTION: Fixation, permeabilization, and staining
?. 
SECTION: Slide preparation
?. 
SECTION: Slide preparation
?.
TITLE: Relative hypotension in emergency care
AUTHORS: James D van Oppen, Lucy Beishon, Rhiannon Owen, Tim Coats

[DESCRIPTION]
Investigation for the effect (on mortality & length of hospital stay) of hypotension at emergency care attendance relative to an individual's baseline

[STEPS]
SECTION: Preparation
2. Dataset generation
SECTION: Preparation
1. Regulatory approvals
SECTION: Data processing
3. Dataset linking and cleaning
SECTION: Data processing
4. Summary statistics
SECTION: Data analysis
5. Regression models
TITLE: m6A visualization/immunofluorescence of DamID
AUTHORS: Lucian Dipeso, Emily Hatch

[DESCRIPTION]
Used for visualizing Dam activity by immunofluorescence in mammalian cells. This protocol stains N-6 methyladenosine (m6A) modified DNA by immunofluorescence while preserving other epitopes, avoiding harsh denaturation steps like heat or chemical treatments.

[STEPS]
SECTION: Protocol
1. Fix cells in 100% MeOH for 10 min at -20 °C
SECTION: Protocol
2. Block, and RNase treat in 2 ug/mL RNase A in blocking buffer (3% BSA, 0.04% TritonX-100, 0.02% sodium azide in PBS) at 37 °C for 30 min
SECTION: Protocol
3. Wash cells 2x in PBS for 5 min at room temperature 
SECTION: Protocol
5. Digest in 50 U/mL DpnI (NEB #r0176) in 1x CutSmart buffer for 30 min at 37 °C
SECTION: Protocol
4. Permeabilize cells in 2% TritonX-100 in PBS for 10 min at room temperature 
SECTION: Protocol
6. Wash 3x in PBS for 5 min at room temperature
SECTION: Protocol
7. Incubate cells in rabbit-anti-m6A (Synaptic, #202 003) diluted 1:500 in PBS for 30 min at room temperature in a humidified chamber
SECTION: Protocol
8. Wash 3x in PBS for 5 min at room temperature 
SECTION: Protocol
9. Incubate cells in anti-rabbit, AlexaFluor conjugated antibody in blocking buffer, diluted according to manufacturer's suggestion, for 30 min at room temperature in a humidified chamber in the dark
SECTION: Protocol
10. Proceed with further immunofluorescence steps and/or DNA staining and mount cells
TITLE: Modified Promega Wizard Extraction for Barcoding Macrofungi
AUTHORS: Stephen       Douglas Russell

[DESCRIPTION]
'This protocol is best used when preparing macrofungal specimens for Sanger sequencing or as a secondary extraction protocol for ONT nanopore barcoding. 

The quality of a DNA extraction method is a primary limiting factor in the total number of samples that will return a result with nanopore barcoding of fungi. The "quick" extraction protocol will often yield a positive result for 80-85% of general fungal collections. Utilizing this extraction protocol pushes that number to nearly 100%. It is more time consuming and utilizes more expensive chemicals, but may be worth considering for important specimens that fail with the quick extraction protocol.

[STEPS]
1. Add 600uL of   to 1.5mL eppi tubes. One tube for each specimen you are planning an extraction for.

2. Place tissue from your specimens into each tube using tweezers. Utilize a piece about the size of a grain of rice. The tissue can be either fresh or dried. Label the tube with the appropriate number. Wipe the tweezers off with a Kimwipe or paper towel in between each specimen. These tubes can be stored at room temperature until they are ready to be used. 
3. Grind the tissue in each tube using a sterile pestle.
4. Heat the tubes at 65 °C for 15 min.

5. Centrifuge the tubes for 3 min.

6. Transfer the supernatant (liquid on top) to a new 1.5mL eppi tube. 

Add 200 µL of   to the tube. 

Vortex the tube for 20 s.

Centrifuge the tube for 6 min.


7. Transfer the supernatant (liquid on top) to a new 1.5mL eppi tube. 

Add 600 µL of 100%   to the tube.  This precipitates the DNA.
 
Centrifuge the tube for 1 min. The DNA will now be in a pellet stuck to the bottom of the tube.

Discard the supernatant. It can just be poured out of the tube into a waste container.

8. Add 600 µL of 70% ethanol to the tube.  

Centrifuge the tube for 1 min.

Discard the supernatant. It can just be poured directly out of the tube into a waste container.

Place the tube upside down on a Kimwipe for at least 15 min, or until all of the ethanol has evaporated from the tube. I usually leave the tube to dry overnight.

9. Add 30uL of molecular water to the tube.

Your DNA template is now ready for amplification.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
Recipe to make 10ml of 2x stock

[GUIDELINES]
<table>
<tbody>
<tr>
<td>1M Tris-HCl pH 6.8</td>
<td>1.0 ml</td>
</tr>
<tr>
<td>SDS</td>
<td>0.4 g</td>
</tr>
<tr>
<td>Glycerol</td>
<td>2.0 ml</td>
</tr>
<tr>
<td>0.5 M EDTA</td>
<td>0.5 ml</td>
</tr>
<tr>
<td>Bromophenol Blue</td>
<td>4 mg</td>
</tr>
<tr>
<td>β-mercaptoethanol (14.7M)</td>
<td>0.2 ml</td>
</tr>
</tbody>
</table>
* It is better to add BME right before using buffer.

[STEPS]