eamag/protocols-with-steps huggingface dataset:
TITLE: RNA extraction from colonial tunicates
AUTHORS: Marta   Wawrzyniak, Simon  Blanchoud

[DESCRIPTION]
This protocol has been successfully used with Botrylloides diegensis and has been adapted from the following publication: 

An efficient low-cost laboratory workflow for the study of blood cells and RNA extractions in marine invertebrates

[GUIDELINES]
Change gloves frequently, particularly as the protocol progresses from crude extracts to more purified materials. Use sterile tubes. Perform all steps on ice and use RNAse-free water unless otherwise stated.

[STEPS]
1. Clean the slide from which you will take the colony of your interest. See Cleaning colonial ascidians.
  
2. Isolate a cleaned colony composed of approx. 20 zooids. 
2.1. Transfer to a tube and spin at maximum speed for 2 min .

2.2. Remove the excess water and shock-freeze the tube in liquid nitrogen. 
3. Add 400 µL of extraction buffer to the frozen sample and macerate with a plastic pestle. 

4. Add 100 µL more of extraction buffer and 500 µL of 1:1 phenol:chloroform.  

5. Mix the tube by inversion a couple of times until it gets cloudy.  
6. Centrifuge the homogenate at 1400 g for 5 min  at 4 °C .

7. Carefully collect 400 µL of the upper phase into a new tube. 

7.1. Note: if desired this sample could be used for DNA extraction - carefully transfer 200 µLof the interphase into a new tube (See DNA extraction from colonial tunicates).  

8. Add 500 µL of 6 Molarity (M) LiCl to the supernatant.

9. Incubate the mixture at -80 °C for 60 min .

10. Centrifuge at 1400 g for 10 min  at 4 °C .
11. Discard the supernatant and resuspend the pellet in 1 mL of 3 Molarity (M) LiCl. 

12. Shake slowly for 15 min at Room temperature on a linear shaker. 

13. Centrifuge at 1400 g for 10 min  at 4 °C .
14. Discard the supernatant and resuspend the pellet in 1 mL of SC-EtOH solution.  

15. Incubate at -80 °C for 15 min .

16. Centrifuge at 1400 g for 15 min  at 4 °C .
17. Discard the supernatant and wash the pellet with 1 mL of 70 % volume Ethanol. 

18. Centrifuge at 1400 g for 5 min  at 4 °C .
19. Discard the supernatant and place the tubes up-side-down on a paper towel for 5 min to 10 min .  

20. Resuspend the pellet in RNase-free water (20 µL to 100 µL depends on the amount of pellet).

22. Store at -80 °C .

21. Quantify the RNA concentration and quality using the NanoDrop, the capillary electrophoresis and/or the Bioanalyzer.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
This tutorial explains how to use VIROME to discover novel genetic polymorphism from viral metagenome shotgun data. <br /><br />Wommack, K. E., J. Bhavsar, S. W. Polson, J. Chen, M. Dumas, S. Srinivasiah, M. Furman, S. Jamindar, and D. J. Nasko. 2012. VIROME: a standard operating procedure for analysis of viral metagenome sequences. Standards in Genomic Sciences 6:427-439 [<a href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3558967/" target="_blank">PMC3558967</a>] <br /><br /><a href="http://virome.dbi.udel.edu/" target="_blank">See site</a> for more information.

[GUIDELINES]
See full video below for more information. <br /><br /><iframe src="https://www.youtube.com/embed/Jj3KP5ri7mY" width="560" height="315" frameborder="0" allowfullscreen="allowfullscreen"></iframe><br /><br />YouTube link: <a href="https://www.youtube.com/watch?v=Jj3KP5ri7mY" target="_blank">https://www.youtube.com/watch?v=Jj3KP5ri7mY</a>

[STEPS]
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TITLE: Ussing chamber experiments for distension evoked secretion in mucosa/submucosa preparations from human colon
AUTHORS: Klaus Michel, Stefanie Schäuffele, Michael Schemann, Gemma  Mazzuoli-Weber

[DESCRIPTION]
This protocol is to investigate the mechanisms of mechanically induced secretion in human colon. For this purpose, we used the voltage clamp Ussing chamber method to gain insights into the distension induced secretion evoked by pressure application to the mucosal or serosal side in the human colon mucosal/submucosal samples. We could show that distension elicited a secretory response due to Cl- and HCO3- fluxes into the lumen. Prostaglandins played an important role in distension induced secretion, because the cyclooxygenase inhibitor piroxicam significantly reduced the distension induced secretion response. Subsequent application of tetrodotoxin revealed a minor, but significant nervous component. This was likely due to activation of mechnosensitive synapses because synaptic blockade by ω-conotoxin GVIA reduced the response to distension. Secretion was induced by tensile rather than compressive forces as preventing distension by a filter inhibited the secretion.

[STEPS]
1. Samples of human colon were placed in ice-cold oxygenated Krebs solution and washed by gentle agitation. Tissues were then dissected in fresh ice-cold oxygenated Krebs solution to obtain preparations containing the mucosa and all submucosal plexi.
2. Tissue samples were cut into pieces of 1cm² or 0.5cm² and mounted in the Ussing chamber holders. The holders were inserted into the Ussing chambers, and the chambers were filled with Krebs solution.
4. As a test for tissue viability, tissue resistance and the response to electrical field stimulation (EFS, 10Hz, pulse duration 1ms, 20V, 10sec) were recorded
3. The tissue was equilibrated for 60min.
5. For the mechanical stimulation both halves of the chambers were sealed with silicon elastomer plugs and the pressure pump was connected to the carbogen gas inlet of the mucosal or serosal chamber
6. Pharmacological substances were added on the serosal or mucosal side 30min before a mechanical stimulation.
7. For analysis, we calculated the integral of the response curve in the LabChart software
TITLE: Site-directed mutagenesis
AUTHORS: Johannes Stuttmann

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Site-directed mutagenesis (SDM) as described here is used to introduce a mutation in the sequence of a plasmid of interest by PCR-based methods. Several different approaches have been previously reported, and also the generation of multiple mutations in a single step is possible. This protocol deals with the simplest version of SDM for production of a small mutation at a single position of a plasmid, </span><span style = "font-style:italic;">e.g.</span><span> for generation of a protein variant or elimination of a restriction site.</span></div></div>

[STEPS]
TITLE: University College Dublin (UCD) Zebrafish Facility Environmental Parameters: 2019
AUTHORS: Grace Kennedy, Husvinee Sundaramurthi, UCD Biomedical Facility Staff , Breandán Kennedy 

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:justify"><span>Environmental factors and animal husbandry conditions are known to have implications in outcome of study results. To this end, environmental parameters (</span><span style = "font-style:italic;">i.e.</span><span> temperature, pH and conductivity) of our zebrafish facility are constantly monitored to ensure constant/limited variations to be introduced in the environment, and hence affecting our research studies. Our facility com. prises of a recirculating water system with filtration, sterilization and automatic adjustment of water parameters (pH, conductivity, temperature) with a capacity of over 4,000 litres of water, it houses over 20,000 adult fish in 1-, 3-, and 10-litre tanks. A separate nursery system houses up to 12,000 larvae aged from 6 to 30 days post fertilisation. </span></div><style>
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							</style></div><div class = "text-block"><div class = "justify" style = "text-align:justify">We have included information of our facility and key parameters measured for our zebrafish lines housed in University College Dublin for the year 2019. These conditions are applicable to all recent peer-reviewed articles published by our group.</div><style>
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[STEPS]
TITLE: Active Absorption of Glucose in the Small Intestine
AUTHORS: Biolprocess

[DESCRIPTION]
This is the protocol for the active absorption of glucose from within the small intestine. This process allows for the absorption of glucose at low concentrations. 

[STEPS]
SECTION: Active Absorption of Glucose in the Small Intestine
1. When the concentration gradient between the blood and the ileum is low, active methods may be used to absorb glucose into the blood
SECTION: Active Absorption of Glucose in the Small Intestine
2. Co-transport proteins in the cell membranes of the cells lining the ileum
SECTION: Active Absorption of Glucose in the Small Intestine
3. Sodium ions are actively pumped out of epithelial cells, into blood
SECTION: Active Absorption of Glucose in the Small Intestine
4. Higher concentration of sodium ions in ileum than in epithelial cells
SECTION: Active Absorption of Glucose in the Small Intestine
5. Sodium ions diffuse down the concentration gradient, into the epithelial cells, through a co-transport protein in the cell membrane
SECTION: Active Absorption of Glucose in the Small Intestine
6. As sodium ions move into the cell, glucose molecules are coupled with them, carrying the glucose up the concentration gradient
TITLE: INTRAMUSCULAR PFF INJECTION PROTOCOL
AUTHORS: LEE LAB RESEARCH LABORATORY, Michael Lee

[DESCRIPTION]
This protocol details the intramuscular PFF injection procedure.

[STEPS]
SECTION: Procedure
1. Clean the procedure hood.
SECTION: Procedure
2. Turn on the heating pad and cover it with a blue procedure pad.
SECTION: Procedure
3. Connect dental needle with tubing.
SECTION: Procedure
4. Fill up PBS into the tubing using a small syringe.
SECTION: Procedure
5. Insert the Hamilton Syringe into the tube for 2-3 mm.
SECTION: Procedure
6. Create air gap between saline solution and PFF solution.
SECTION: Procedure
7. Keep mouse inside desiccator w/ isoflurane until breath becomes long and slow.
SECTION: Procedure
8. Two separate people should inject each respective Bicep femoralis of the mouse simultaneously.
SECTION: Procedure
9. Perform injection with animal on warm pad and keep clean recovery cage on separate heating pad until animal recovers.
SECTION: Procedure
10. Wipe needle with alcohol wipes between sample draw/mouse injection.
TITLE: Slice Preparation and Blockface Imaging for Electrophysiology - Mouse
AUTHORS: Allen Institute for Brain Science

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes the procedure for transcardial perfusion of an adult mouse, followed by dissection, embedding, slicing, and blockface imaging of the mouse brain using either a Leica vibratome or a compresstome. The output tissue is suitable for use in electrophysiological recordings and/or live cell filling applications.</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 U19MH114830. 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: Purification of NIX-GST
AUTHORS: Elias Adriaenssens

[DESCRIPTION]
This protocol details the purification of NIX-GST.

[STEPS]
SECTION: Purification
1. To purify NIX-GST, fuse the cytosol-exposed domain of NIX (1-182aa) to a C-terminal GST-tag through cloning into a pET-DUET1 vector (available from Addgene). 
SECTION: Purification
2. Introduce the point mutants in vitro mutagenesis to generate NIX E72A/L75A/D77A/E81A (4A; ΔWIPI2) (available from Addgene), and NIX W35A/L38A (ΔLIR) (available from Addgene).
SECTION: Purification
3. After the transformation of the pET-DUET1 vector encoding NIX-GST wild-type or mutants in E. coli Rosetta pLysS cells (Novagen Cat# 70956-4), grow the cells in 2x Tryptone Yeast extract (TY) medium at 37 °C until an OD600 of 0.4 and then continue at 18 °C. 
SECTION: Purification
4. Once the cells reaches an OD600 of 0.8, induce the protein expression with  100 micromolar (µM) isopropyl β-D-1-thiogalactopyranoside (IPTG) for 960 min at 18 °C. 
SECTION: Purification
5. Collect the cells centrifugation and resuspend in lysis buffer. 

 Lysis buffer:
 
 
SECTION: Purification
6. Sonicate the cell lysates twice for 30 s and clears by centrifugation at 18.000 rpm, 45 min, 4 °Cin a SORVAL RC6+ centrifuge with an F21S-8x50Y rotor (Thermo Scientific). 

SECTION: Purification
6.1. Sonicate the cell lysates for 30 s (1/2). 

SECTION: Purification
6.2. Sonicate the cell lysates for 30 s (2/2). 

SECTION: Purification
7. Collect the supernatant and incubate with pre-equilibrated Glutathione Sepharose 4B beads (GE Healthcare) for 120 min at 4 °C with gentle shaking to bind NIX-GST. 
SECTION: Purification
8. Centrifuge the samples to pellet the beads and remove the unbound lysate. 
SECTION: Purification
9. Wash the beads twice with wash buffer, once with high salt wash buffer, and two more times with wash buffer. 

 Wash buffer: 

 

High salt wash buffer:
 
 
 
SECTION: Purification
10. Incubate the beads  120 min with4 mL of 50 millimolar (mM) reduced glutathione dissolves in wash buffer at 4 °C, to elute NIX-GST from the beads. 

 Wash buffer: 
 
 
 
SECTION: Purification
11. To collect the supernatant, collect the beads by centrifugation. 
SECTION: Purification
12. Wash the beads twice with 4 mL of wash buffer, and collect the supernatant. 
SECTION: Purification
13. Pool the supernatant fractions, filter through a 0.45 µm syringe filter, concentrate with 10 kDa cut-off Amicon filter (Merck Millipore), and load onto a pre-equilibrated Superdex 200 Increase 10/300 GL column (Cytiva). Elute the proteins with SEC buffer. Analyze the fractions by SDS-PAGE and Coomassie staining. Pools fractions containing purified NIX-GST. 

 SEC buffer:

 
 
SECTION: Purification
14. After concentrating the purified protein, aliquote the protein and snap-frozen in liquid nitrogen.
TITLE: Protocol for a scoping review to map the evidence around laboratory tests for use in low resources settings
AUTHORS: Virginie Viprey, Kerrie Davies, Jane Freeman, Robyn Meurant, Mike Messenger

[DESCRIPTION]
The aim of this thematic evidence review is to identify and provide
guidance on the application of different laboratory tests modalities with a
focus on Low- or Middle-Income Country (LMIC)

[STEPS]
TITLE: Immunofluorescence of ATP13A2 and CD63
AUTHORS: Stephanie Vrijsen, Peter Vangheluwe

[DESCRIPTION]
This protocol describes the seeding, fixation and staining of samples for immunofluorescent visualization of ATP13A2 and CD63. 

[STEPS]
1. Seed cells: 75 000 cells/well (SH-SY5Y) in a 12-well plate with coverslips. Allow the cells to attach and grow for 2880 min.

2. Wash the cells with ice-cold PBS.
4. Wash the cells twice with ice-cold PBS. 
5. Permeabilization and blocking with a mixture of 5% BSA (Roth) and 0.5% saponin (Sigma) (diluted in PBS) (referred to as blocking buffer) for 60 min on a shaker.

6. Incubate with primary antibody (anti-ATP13A2, A3361, Sigma; anti-CD63, 11-343-C100, ExBio) (diluted 1/200 in blocking buffer) for 120 min (40 µl antibody mixture is sufficient per coverslip in a wet chamber). 

8. Incubate the samples in secondary antibody (alexa-488 goat anti-rabbit IgG R37116, Invitrogen; alexa-594 goat anti-mouse IgG, A11005, Invitrogen) (diluted 1/1000 in blocking buffer) for 30 min on a shaker.

10. Incubate the samples in 200 ng/ml DAPI (D9542, Sigma) for 15 min . 

12. Mount the samples with FluorSave (345789-20ml, Merck). 
3. Fix the cells with 4% paraformaldehyde 30 min at 37 °C. 

7. Wash with DPBS for 5 min on a shaker, repeat this step 3x. 

9. Wash with DPBS for 5 min on a shaker, repeat this step 3x. 
11. Wash with DPBS for 5 min on a shaker, repeat this step 3x. 
13. Acquire images using a LSM880 microscope (Zeiss) with a 63x objective and Airyscan detector.
TITLE: &#34;Reward perseveration is shaped by GABAA-mediated dopamine pauses&#34;, Burwell et al
AUTHORS: Sasha Burwell, HaiDun, MICHAEL R TADROSS

[DESCRIPTION]
This collection of protocols details the experiments and analysis performed for the Burwell et al paper "Reward perseveration is shaped by GABAA-mediated dopamine pauses". Software used in the various analysis protocols can be found at https://github.com/tadrosslab/VTA_GABA_paper.

[STEPS]
TITLE: FloodLAMP Binding Solution Prep v1.1
AUTHORS: Randy True

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The Binding Solution includes NaI, HCl, Triton-X100, and water. We have had lots of trouble with it, largely due to a mistake that has been corrected. In our hands currently, the Binding Solution is a pale yellow—a possibility mentioned in the Rabe Cepko paper. It does seem to become cloudy over time (days to weeks). Our current plan is to prepare weighed-out NaI and the other components premixed (stored at 4C) for easier prep, and then make fresh Binding Solution up at least weekly.</div><div class = "text-block">We have moved to storing the Binding Solution in 5mL amber screw cap tubes for development runs (2.25mL for a single strip of 8 samples), with these in an opaque container. This makes it convenient to add glass milk and use it as a master mix (as suggested by Rabe Cepko). Care must be taken to keep the silica resuspended prior to pipetting for the addition to the sample, hence the mixing by pipetting on every draw.</div></div>

[STEPS]
?. Weight out 45g of NaI into 50mL Falcon Tube
?. Add 1st traunch of water to 45ml
?. Vortex, shake and then vortex again until fully mixed
?. Add 2nd traunch of water to 46mL
?. Vortex, shake and then vortex again until fully mixed
?. Vacuum filter (or wait and vacuum filter many tubes at the end)
?. Add .5mL of 1N HCl
?. Vortex for 10s, be careful that the tube is completely closed
?. Add 1.0 mL of Triton X-100, note that it is very viscous (use 1000ul tip with end cut off)
?. Vortex for 10s and shake
?. Add 2.5mL UltraPure dH2O
?. Vortex 10s
?. Aliquot to 5mL amber Eppendorf screw caps, 2.25mL for 8 x .5mL samples, store in dark box
TITLE: Modified Organic Extraction Protocol (Pedersen et al., 2016)
AUTHORS: Mikkel Pedersen

[DESCRIPTION]
A modified version of the organic extraction protocol used in Wales et al., 2014. 

Protocol successful at reconstructing the biodiversity of 15 kya sediment core, including a few fish species

[BEFORE_START]
Review the manufacturer’s Safety Data Sheet and additional chemical information. Ensure that a written experimental protocol including safety information is available. 

[GUIDELINES]
Phenol should be handled with care as it is corrosive and toxic. 
Chloroform is toxic if swallowed or inhaled. It can cause severe and irreversible health effects, including death

[STEPS]
SECTION: Sample preparation &amp; cell lysis
1. ADD 2 g of sediment sample to a sterile spin tube

ADD 170 µg of proteinase K to the sample

ADD 3 mL of lysis buffer to the sample

VORTEX sample at max speed for 40 s 
 

SECTION: Sample preparation &amp; cell lysis
2. ADD an additional 170 µg of proteinase K to each sample

INCUBATE    at 37 °C
SECTION: Inhibitor removal
3. REMOVE PCR inhibitor using MOBIO (MO BIO Laboratories, Carlsbad, CA) C2 and C3 buffers following the manufacturer’s protocol

 

CENTRIFUGE samples at 10000 x g for 3 min 

TRANSFER supernatant to a sterile microcentrifuge tube

SECTION: Phenol:Chloroform extraction
4. ADD equal volume of Phenol: Chloroform: Isoamyl alcohol (25: 24: 1) solution to samples

ROTATE samples gently at Room temperature for 10 min 

CENTRIFUGE at 3200 x gfor 5 min

DISCARD supernatant
SECTION: Phenol:Chloroform extraction
5. REPEAT step 4

TRANSFER supernatant to a Millipore 15 mL Amicon Ultra filter for concentration and purification
SECTION: DNA concentration and purification
6. CENTRIFUGE filter at 4000 x g for 10 min 

DISCARD liquid flow-through
SECTION: DNA concentration and purification
7. ADD 500 µL EB Buffer (Qiagen) to the filter

CENTRIFUGE at 4000 x g for 10 min 

DISCARD liquid flow-through
SECTION: DNA concentration and purification
8. REPEAT step 7
SECTION: DNA concentration and purification
9. ADD 50 µL molecular grade water to the filter

INCUBATE at Room temperature for 10 min 

CENTRIFUGE at 4000 x g for 10 min 

DISCARD filter

DNA is now ready for downstream applications
TITLE: Overview of scRNA-seq of Human Knee Articular Cartilage
AUTHORS: molmer  , Martin  Lotz

[DESCRIPTION]
The overview provides sub-protocols for each step required to complete scRNA-seq from human knee articular cartilage.

[STEPS]
1. Tissue Acquisition
dx.doi.org/10.17504/protocols.io.14egn7996v5d/v1 
2. Tissue Quality Control
dx.doi.org/10.17504/protocols.io.n2bvj695nlk5/v1
3. Single-Cell Isolation
dx.doi.org/10.17504/protocols.io.14egn765qv5d/v1
4. cDNA Library Preparation
dx.doi.org/10.17504/protocols.io.bp2l6175rvqe/v1
5. Data Processing and Quality Control
 dx.doi.org/10.17504/protocols.io.j8nlkk3q6l5r/v1
TITLE:  Binding  of avian immunoglobulins to peroxidase-labeled anti-chicken IgY conjugate by double immunodiffusion (Ouchterlony) technique.
AUTHORS: Angel  Justiz-Vaillant

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">In this experiment anti-chicken IgY developed in rabbit cross-reacts with a large panel of avian species including ostrich, duck, pigeon, and quail. </div></div>

[STEPS]
?. The binding of  avian immunoglobulins to a secondary antibody rabbit anti-chicken IgY antibody was investigated by the Ouchterlony technique.
?. Briefly, 1% agarose gels are prepared and wells cut into the gel using a template.
?. Initially, aliquots of 25 µl each of rabbit anti-IgY antibody at 1 µg/µl are applied to the centre well.
?. The peripheral wells are filled with 25 µl each of IgY (30 µg/µl) of various avian immunoglobulins Y (IgY) including duck, pigeon, chicken, bantam hen, ostrich, pheasant,guinea hen, goose, quail, cattle egret and other species. This IgY were isolated from the egg by the method of Polson, 1990.
?. The gels are incubated at RT for 48−72 hours on a humidity chamber.
?. After that the gels are examined for precipitin lines.
?. Chicken IgY is included as a positive controls.
?. The positive results are taken as the presence of precipitin line/s and negative results, the absence of precipitin lines.
TITLE: Dialysis using D-Tubes
AUTHORS: Neilier Junior

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Sample preparation is crucial for successful biomolecule analysis. For this, the method must be individually chosen, based on the characteristics of the sample and the analyte. How some strategies can compromise the integrity of the molecule of interest. The dialysis D-tubes allow the concentration of samples with no precipitation by organic solvents or salts.</div></div>

[STEPS]
?. [Material preparation]
Choose the cutting mass (3.5 to 14 kDa) of the dialysis D-Tube based on the biomolecule to be purified.
?. [Procedure]
Complete the dialysis D-Tube with Milli-Q water and let it equilibrate for 15 min
?. [Procedure]
Remove the water and weigh the dialysis tube
?. [Procedure]
Add the sample with the aid of a pipette and weigh the tube again
?. [Procedure]
Prepare a beaker with 1000 mL of Milli-Q water (or exchange buffer) and a magnetic stir bar
?. [Procedure]
Place the dialysis tube on a floating rack and immerse the tube in the water in the beaker
?. [Procedure]
Cover the beaker with plastic film, start stirring and monitor the conductivity of the solution in the beaker. When the conductivity stops increasing, the concentrations between the sample and the solution are in equilibriumIf conductivity is not controlled: 1 h of dialysis suffices to desalinate 3 M of ammonium sulfate in these conditions
?. [Procedure]
Remove the dialysis tubes from the beaker and weigh the contents