eamag/protocols-with-steps huggingface dataset:
TITLE: Experiment protocol: a syringe-filter based DNA extraction
AUTHORS: Sungwoo Bae

[STEPS]
?. Prepare the following solutionsa. TE buffer (10 mM Tris-HCl and 1 mM EDTA)b. TE/lysozyme (TL) buffer (10 mM Tris-HCl, 1 mM EDTA, and 7.5 mg/mL lysozyme)c. TE/proteinase K/SDS (TPS) buffer (10 mM Tris-HCl, 1 mM EDTA, 300 µg/mL proteinase K, and 1% SDS[w/v])
?. Filtration The 10 mL sample or 100ml  was passed through a 0.2 µm pore-sized syringe filter in the direction of the filtration.
?. TE buffer addition A 1 mL of TE buffer was injected into the same filter, still in the direction of the filtration; the flow through was pooled into a sterile 15 mL tube.
?. Backwasing stepBackwashing was done in the opposite direction of filtration with the flow through pooled into the same 15 mL tube; the tubes were mixed by inversions for 5 mins in between each back-washing step:a. A 1 mL of TL buffer was flushed through the filter then 300 µL of TL was added into the pooled flow-through.b. Next, 1 mL of TPS buffer was flushed through the filter.c. Lastly, 1 mL of TPS buffer and 3 times of 1 mL air was flushed and mixed to the flow through.d. The filter is then discarded
?. Magnetic beads addition  A 50 µL of silica-coated magnetic beads solution (Dynabeads MyOne SILANE, Invitrogen) was added to the flow-through and was mixed by inversions for 5mins.
?. Washing Optional: Washing buffer (1 mL) (10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0, 70% ethanol) was added and mixed by pipetting the mixture several times. The beads were magnetized to the tube walls then the supernatant was discarded.
?. DNA elution and collectiona. The DNA was then eluted from the beads with 50 µL of TE buffer.b. The magnetic beads were collected, and the DNA samples were recovered.c. The recovered DNA was then diluted 3x with TE buffer to lessen possible inhibitions on downstream applications. The diluted DNA can also be stored in -20oC.
TITLE: Mouse model of post-colitis (DNBS) chronic visceral hypersensitivity.
AUTHORS: Andrea   Harrington

[DESCRIPTION]
This protocol is used to administer DNBS (Dinitrobenzene sulfonic acid) to the colorectum of mice via intracolonic enema administration to induce mild colonic inflammation.

[STEPS]
1. Fast mice overnight by removing bedding and food from cages and placing a wire fasting rack at the bottom of cage. Provide 5% glucose drinking water. Record weight before fasting.
2. Weigh mouse following overnight fasting, if mouse has lost more than 10% of prefast weight, or is under 22 grams do not treat animal, rehouse in a cage with fresh bedding, free access to food and water.
3. Weigh out the desired amount of DNBS to achieve a concentration of 6.5mg/ml and in a volume enough to administer 0.10mL /mouse. Dissolve DNBS in the desired volume of 35% ethanol. Make sure to wear gloves during handling of DNBS and weigh in an appropriate Fume Cupboard.
4. Load a 1 ml syringe with 0.10mL DNBS. Attach a 3cm cannula to syringe and remove dead space.
5. Place mouse in induction box. Induce anesthesia with 5% isofluorane, 2% oxygen. Once the mouse is fully anaesthetized (absence of pedal reflex upon pinch) lower isofluorane to 1.5 % for maintenance. 
6. Within induction box, place animal head down into a 50 mL syringe so that the mouse receives anesthetic whilst the lower trunk and rectum is accessible. Check pedal reflexes to ensure mouse is deeply anaesthetized.   
7.  Lubricate both the mouse rectum (KY Jelly, Durex) and the sides of the cannula and slowly insert the cannula into the rectum until it is 3 cm from the mouse rectum opening. Take care not to puncture the colon wall. If resistance to cannula insertion is felt, stop insertion and take note of the depth inserted. Resistance may be due to some residual faeces remaining in the colon even though the mouse has been fasted overnight, which may make it difficult to move the whole 3 cm.
8. Very slowly inject 0.10mL of DNBS solution as slowly withdrawn cannula out of the colorectum. As the cannula is withdrawn, the DNBS solution will gradually leak out of the cannula and not expose past the targeted area. 
9. Remove mouse from tube and hold vertically for 30 seconds to prevent leakage of instilled DNBS due to gravity and allow uniform colorectal exposure. 
11. Post-DNBS instillation mice are to be housed individually with free access to food and 5% glucose water, soaked food and sunflower seeds. Cages are to be placed on heat pads for 2-3 days post-DNBS instillation.
12. Monitor mice (including weigh) at least twice per day for 7 days post-DNBS enema as per approved post procedure monitoring requirements. Monitor and record for score severity of colitis (and related discomfort), such as weight loss, diarrhoea, constipation, blood in stools, GRIMACE (pain score), lack of movement and grooming (hunched/ruffled) 
10. Place mouse into a new cage with clean bedding and monitor recovery until anesthetic wears off and mouse is ambulatory.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol is provided to synthesize single-stranded cDNA from total RNA samples using the TaqMan® MicroRNA Reverse Transcription Kit and quantification of miRNA using the comparative Cycle threshold (Ct) method. Endogenous controls are used to normalize the expression levels of target miRNAs by correcting differences in the amount of cDNA loaded into the PCR reactions. An appropriate constitutively expressed endogenous control must be selected.</p>

[GUIDELINES]
<p>For the optimal performance of the TaqMan MicroRNA Reverse Transcription Kit and of the TaqMan MicroRNA Assays, use RNA with the following characteristics:</p>
<ul>
<li>Free of inhibitors of reverse transcription and PCR,</li>
<li>Dissolved in PCR-compatible buffer;</li>
<li>Free of RNase activity;</li>
<li>Allow the kit components to thaw on ice.</li>
</ul>

[STEPS]
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TITLE: Assessment of the impact on future risk of diseases associated with low birth weight: overview of reviews
AUTHORS: Yoshiko Yamamoto, Yasushi Tsujimoto, Shunsuke Taito, Yusuke Tsutsumi, Yoshitaka  Wada, Yasutaka Kuniyoshi, Takashi  Ariie, Daishi Hirano, Masahiro Banno

[STEPS]
TITLE: Dual coloMOCA implantation protocol 
AUTHORS: Brett Hanzlicek, Dennis Bourbeau

[DESCRIPTION]
This protocol describes the implantation of two  bowel devices (ColoMOCA) in pigs.

[STEPS]
SECTION: Anesthesia
1. Anesthetize/prep the animal for surgery.

       Aseptic procedures by trained personnel are utilized during the surgery. All tools are sterilized         prior to insertion. Drapes will be used and personnel performing surgery will wear clean surgical scrubs, and sterile gowns/gloves and masks.
SECTION: Laparotomy
2. Make a midline suprapubic incision ~9 cm long to expose the peritoneal cavity and viscera.
SECTION: Colotomy
3. Make two small incision into the colon.
SECTION: Insert and anchor ColoMOCA
4. Insert devices through the small incision and secure in place with sterile non-absorbable sutures.
SECTION: Test Wireless Transmission
5. Wake the coloMOCAs with radio if not already awake.  Confirm data transmission.
SECTION: Close incision
6. Close the colon with an absorbable suture in an interrupted pattern
Close the peritoneum and fascia with absorbable sutures using a continuous suture pattern.
Close the subcutaneous tissue and skin with non-absorbable monofilament suture with a cutting needle, using an interrupted suture pattern. Skin staples may also be used.
SECTION: Test Wireless Transmission (2)
7. Confirm data transmission and then put devices to sleep
SECTION: CT-Imaging
8. Image to verify placement of the sensors.
SECTION: Place jacket on the pig
9. Place and zip the jacket on the pig while still anesthetized.
SECTION: Wake up the animal
10. Stay with the animal until fully awake.  Return to pig room.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p><strong>This is a short tutorial on how to get started analyzing FASTA files via the command line.</strong></p>
<p> </p>
<p>Code is intended for use on an Ubuntu 16.04 LTS OS, but it may work on other Unix or Unix-like systems.</p>
<p> </p>
<p>Here we will use the ETE3 toolkit, which is a very nice tool for phylogenetic reconstruction. The main website is here: http://etetoolkit.org/</p>
<p>We will also look at the EggNOG database, which is a very useful database of protein families: http://eggnogdb.embl.de/#/app/home</p>
<p> </p>
<p> </p>

[STEPS]
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TITLE: UPitt TriState SenNet TMC Lung Procurement
AUTHORS: John Sembrat, Alison Morris, koenigshoffm, Oliver Eickelberg

[DESCRIPTION]
This document outlines the request for specimen procurement before arrival at the TriState SenNet TMC Biospecimen Core at the University of Pittsburgh, as part of the Cellular Senescence Network Program (SenNet).

[STEPS]
SECTION: Requested processing protocol for Donation following Brain Death Declaration (DBD)
1. Organ Procurement Organization Recovery Team prepares for organ recovery as consistent with organ transplant protocol with cross clamp and in situ cold flush body cooling with minimal warm ischemic time.

Includes flush with Perfadex or similar organ transplant buffer, until solution runs clear
SECTION: Requested processing protocol for Donation following Brain Death Declaration (DBD)
2. Complete a minimum back-table lung flush with transplant buffer of 500ml into the pulmonary artery and 500ml into the pulmonary vein. 

Do not flush with saline.
Do not wrap in surgical towel.
SECTION: Requested processing protocol for Donation following Brain Death Declaration (DBD)
3. Staple, clamp, or tie the trachea closed prior to shipping. 
SECTION: Requested processing protocol for Donation following Brain Death Declaration (DBD)
4. Place Lung(s) in a Specimen Bag and submerge with chilled Perfadex or similar. Triple bag the tissue.
SECTION: Requested processing protocol for Donation following Cardiac Death /Arrest(DCD)
5. Organ Procurement Organization / Recovery Team prepares for organ recovery as quickly following Cardiac Arrest while in comfort care as authorized by next of kin.
SECTION: Requested processing protocol for Donation following Cardiac Death /Arrest(DCD)
6. Remainder of organ / tissue recovery should be as consistent with organ transplant protocol (section above) as possible.

Includes organ flush with Perfadex or similar organ transplant buffer until solution runs clear.
SECTION: Requested processing protocol for Donation following Cardiac Death /Arrest(DCD)
7. All warm ischemic time is to be minimized and reported. Tissues may be accepted up to 6 hours WIT.  Other WIT may be evaluated depending of the circumstances.
TITLE: Sinai SCENT TMC - 5x5 Project Bulk RNAseq
AUTHORS: HIMC at Mount Sinai

[DESCRIPTION]
Bulk RNA sequencing protocol

[STEPS]
SECTION: Protocols
1. Dissect the snap-frozen or -80'C stored tissues.
SECTION: Protocols
2. Dissected tissues are placed directly into a labeled 1.5 mL RNAlater (Qiagen) collection tube and stored at 4°C for up to 24 hours to allow the RNAlater to penetrate the tissue fully before being transferred to -20°C or lower until use. 
SECTION: Protocols
3. The mRNA is extracted with the RNeasy Plus Mini kit (Qiagen) per the manufacturer’s instructions.
SECTION: Protocols
4. The complementary DNA (cDNA) library is established by using the Ovation RNA-Seq v2 method (NuGen). 
SECTION: Protocols
5. Samples are sequenced on an Illumina NovaSeq sequencer with 2X150 bp in the paired ends at 350M read depth per sample.
SECTION: Protocols
6.  Gene and isoform expression levels will be calculated using Cufflinks.
SECTION: Protocols
7. Transcripts from mitochondrial and ribosomal RNA genes are masked and not included.
SECTION: Protocols
8. The final abundances of genes in cells are computed in FPKM (Fragments Per Kilobase of exon model per Million mapped fragments).
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This week we're going to take a closer look at the taxonomy of your datasets using mothur. We'll classify taxonomy and diversity using the metagenomic reads that mapped to 16S ribosomal RNA. The Tara Oceans folks have already pulled out the reads that match 16S ribosomal RNA, so we're going to use those datasets.</p>

[STEPS]
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TITLE: Citric Acid Water Restriction
AUTHORS: Thomas R Clarke

[DESCRIPTION]
How to prepare and carry out citric acid water restriction for motivating mice to engage in behaviour.

[STEPS]
SECTION: Baseline Weights
1. Handle animals and weigh for 3-5 days to establish a mean baseline weight prior to introduction of the CA water
SECTION: Preparing 2% CA Water
2. Prepare the following:

2 bottles
Citric acid crystals
Sterile Gloves or weighing boats
Scales
SECTION: Preparing 2% CA Water
2.1. Empty one of the water bottles, place it on the scales and tare/zero
SECTION: Preparing 2% CA Water
2.2. Pour the water from the other bottle into the one on the scales, record the weight of water
SECTION: Preparing 2% CA Water
2.3. Calculate 2% weight of the water, this is the weight of citric acid crystals you need

e.g. 300g of water, requires 6g of citric acid (300*0.02 = 6)
SECTION: Preparing 2% CA Water
2.4. Use the plastic side of the packet containing the sterile gloves or a weighing boat to weigh out the citric acid crystals
SECTION: Preparing 2% CA Water
2.5. Add crystals to the bottle of water, put on the lid/spout and shake with finger covering the opening until dissolved
SECTION: Preparing 2% CA Water
2.6. Label the bottle on 2 sides as "2% Citric Acid". Add a 'DO NOT REMOVE' label to the base of the bottle
SECTION: Starting water restriction
3. Replace normal water bottle with 2% CA bottle, perform behaviour with 10% sucrose as a reward
SECTION: Starting water restriction
3.1. Weigh animals daily to ensure they do not drop below 80% of their baseline weight


  

SECTION: Starting water restriction
3.2. If any animal drops below 80% of their baseline weight, return to ad lib water per the PPL
TITLE: Sample Site Questionnaire
AUTHORS: Dilip  Abraham, Nicola Elviss, Nicholas Feasey, Nick  Grassly, Jacob John, Gagandeep Kang, John  Scott Meschke, Venkata  Raghava Mohan, Satheesh Nair, Jonathan  Rigby, Rajan Srinivasan, Catherine  Troman, Christopher  Uzzell

[DESCRIPTION]
The following document is a questionnaire for recording sampling site details. The first set of questions is for details that should be recorded every time a sample is collected from that site. The second set is to be completed once for each site to give more general details.

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>A protocol to create a genomic library (2-5 kb insert size) to screen for genomic regions functioning as centromeres. Uses the NEBNext Illumina library prep kit from NEB to add adapters to fragments and then the fragments can be efficiently assembled into a vector containing homology to the adapter sequences using Gibson Assembly.</p>

[STEPS]
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TITLE: Two-action sequence reinforcement 
AUTHORS: Jonathan Tang

[DESCRIPTION]
Two-action sequence reinforcement protocol for mouse studies from Tang et al 2023. 

[GUIDELINES]
Individual mice were subjected to a single session of protocol each day, with sessions following each other on consecutive days. 

[STEPS]
SECTION: First reinforcement session+ Baseline
1. Mice were placed in a grey open-field behaviour recording for this protocol. 
To acquire baseline behavior, individual mice were allowed to behave freely inside the box for 30 minutes when the laser stimulation was not available for reinforcement. 
2. After initial behavior acquisition, 60 minutes of laser availability was given. During the
reinforcement periods, when the closed-loop system detects performance of the
proximal action (T1) of interest, the algorithm enters a state in which the
laser is triggered on performance of the distal action (T2), regardless of the
amount of time that has elapsed between the latest T1 and T2. 
SECTION: Remaining Sessions
3. 90 minutes of closed loop reinforcement were made available for individual mice during subsequent sessions.
During the reinforcement periods, when the closed-loop system detects performance of the
proximal action (T1) of interest, the algorithm enters a state in which the
laser is triggered on performance of the distal action (T2), regardless of the
amount of time that has elapsed between the latest T1 and T2. 
SECTION: Extinction protocol
4. An extinction protocol was carried out comprising of 20-minute maintenance of reinforced behavior with laser availability, followed by 60 minutes of extinction of reinforced behavior without laser availability, followed by 20-minute re-acquisition of reinforced behavior with laser availability.
TITLE: Euplotes crassus transfection using Lipofectamine 2000 as vehicle (provisional)
AUTHORS: RACHELE CESARONI, Rachele Cesaroni

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

[STEPS]
?. Collect 4 x 104 well-fed Euplotes crassus cells (we used E.coli as the only food source) by centrifugation at 400 rcf for 3 minutes.
?. Wash the cells twice with artificial sea water (see attachment for the recipe) and once with 500 mM sorbitol, 0.5 mM Tris-HCl, pH 7.0 (400 rcf for 3 minutes each time). Then resuspend Euplotes crassus cells in 50 μl of the medium (500 mM sorbitol, 0.5 mM Tris-HCl, pH 7.0). To get this small volume you may require an additional minute of centrifugation.
Recipe for complete sea water (1 L):

36 g Reef Crystals
1 ml Walne's solution
1 ml of 10 μg/ml FeSO40.2 ml of 2 mg/ml thiamine (light sensîtive; store at 4 ºC)
Add distilled water up to 1 L
?. Transfer the cells into wells within a 96-well plate for transfection.
?. Dilute 2.5 μl of Lipofectamine 2000 Reagent in 25 μl of the same medium of the cells (500 mM sorbitol, 0.5 mM Tris-HCl, pH 7.0).
?. Dilute 5 μg of DNA dissolved in MilliQ H2O (0.5-5 μg/μl) in 125 μl of the same medium of the cells (500 mM sorbitol, 0.5 mM Tris-HCl, pH 7.0).
?. Add 25 μl of the diluted DNA to 25 μl of the diluted Lipofectamine 2000 Reagent (1:1 ratio), and incubate them for 10 minutes at room temperature (before incubation mix them by pipetting up and down 5 times or vortex them for 10 seconds).
?. Add 10 μl of the transfection complexes drop-wise to the 50 μl of Euplotes crassus cells in medium (gently swirl the dish to ensure uniform distribution of the transfection complexes).
?. One hour after addition of Lipofectamine 2000 complexes, add 50 μl of artificial sea water to the cells.
?. After another hour, add other 50 μl of artificial sea water to the cells.
?. An hour later harvest the cells (400 rcf for 3 minutes), and wash them twice with artificial sea water (400 rcf for 3 min each time). Then resuspend them in 400 μl of the artificial sea water.
?. Transfer the cells into depression wells.
?. Incubate the cells at 24°C, then analyze them by fluorescence microscopy to determine gene expression.
TITLE: Protein structure prediction with AlphaFold-Multimer
AUTHORS: Elias Adriaenssens

[DESCRIPTION]
This protocol describes the protein structure prediction with AlphaFold-Multimer.

[STEPS]
1. Protein sequences were downloaded from the Uniprot server.   
2. A locally installed version of AlphaFold-Multimer was used for structure prediction with 5 models per prediction followed by Amber relaxation.
3. Interaction scores (ipDT) and diagnostic plots (PAE plot and pLDDT plot) as well as the generated structures were manually inspected.
4. Predicted structures were visualized with ChimeraX-1.8.
TITLE: Chromatographic profile of aqueous extract by HPLC
AUTHORS: Luis Wiliunfo Torres Tapia

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This chromatographic detection method was developed in order to solve the most compounds from aqueous extracts leaves of dune and mangrove plant by HPLC.</div></div>

[STEPS]
?. [Sample preparation]
Each aqueou extract was added a 10% MeOH to improve solubility, and next it was filtered through 0.2 μm filter disc.
?. [Movil phase]
Movil phase consist:-  Movil phase A contaned ultrapure type 1 water ( Simplicity® Water Purification System, Millipore) adjusted to pH 2.5 with trifluoroacetic acid (TFA).-  Movil phase B contained acetonitrile (ACN).Each movil phase was filtered through 0.2 μm filter and finally was degassered during 30 min. in ultrasonic equipment.
?. [Equipment]
-  Profile was carried out on an Agilent Series 1290 Infinity HPLC System with vacuum degasser, quaternary pump, autosampler, thermostated column compartment and photodiode array detector (DAD).-  Data analysis was performed with Agilent HPLC EZChrom software.
?. [Column]
Separation of analytes was performed on an Grace Alltima C18 column (4.6 mm × 250 mm, 5 μm), which was maintained at 35 °C during the analysis.
?. [Elution gradient]
The gradient was programmed as follows: 0-3 min, 5% B; 3-43 min, 5-30% B; 43-73 min, 30-85 %B, 73-75 min 85-5 %B.
?. [Elution flow]
Set the flow rate to 1 mL/min.
?. [DAD monitoring]
Set the DAD monitoring to 254 nm and 350 nm.
?. [Injection]
Inject 20 μL of the sample.
TITLE: Poly-Lysine Coverslip Preparation
AUTHORS: Franchesca Farris, Marda Jorgensen

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Poly-Lysine Coverslip Preparation </div><div class = "text-block">This section describes the process of creating Poly-lysine-coated coverslips that are used for the tissue slices in the CODEX® experiment workflow.</div></div>

[STEPS]
?. Remove the coverslips from box.
?. Gently place coverslips at the bottom of the glass beaker
?. Slowly swirl the beaker to spread the stacks of coverslips.
?. Add ca. 7 mL of poly-lysine solution above the coverslips to ensure that all coverslips are fully covered.
?. Cover the beaker with plastic wrap and seal with a rubber band to prevent evaporation.
?. Leave coverslips in poly-lysine solution for a minimum of 12 hours and up to one week at room temperature.
?. After incubation period, the washing and storing coverslips steps begin.
?. Gradually pour poly-lysine solution into the proper waste container.
?. Fill the beaker containing the coverslips to half volume with ddH2O
?. Swirl the contents to mix the solution.
?. Let the beaker and coverslips sit for 30 seconds.
?. Slowly pour off the water into the sink
?. Repeat steps 9-12 more times for a total of 7 washes.
?. Place two sets of paper towels on the bench top.
?. Remove the coverslips from the water, placing them on top of the first set of lint free paper towels. Ensure coverslips are not overlapping to allow proper drying.
?. Invert each coverslip. Dry the reverse side on the second set of paper towels.
?. Leave the coverslips on the paper towels to dry.
?. When they are dry, the Poly-lysine-coated coverslips can be stored in a petri dish or covered similar container.
TITLE: Crystallization of WIPI2d10-364delta263-295
AUTHORS: lmstrong 

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Setting crystal trays</div></div>

[STEPS]
?. Mix 1uL of reservoir and 1uL of protein solution on coverslip. Add 0.3uL of seed stock to drop.
?. Quickly invert coverslip onto pre-greased 24well plate (Hampton). Gently press on edges of the coverslip to create a good seal
?. Repeat for every well in the plate
?. Let plate sit at 19C. Checking ever 12-24 hours for crystal growth.
?. Once crystal form, make up a solution of the reservoir in 25% glycerol (v/v). Add 1uL of this to a new coverslip. Gently fish the crystal from the drop and soak the crystal in the glycerol solution for 1 minute. Quickly transfer crystal into liquid nitrogen cooled puck for long term storage.