eamag/protocols-with-steps huggingface dataset:
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol can be used to dissociate adult human kidney “on ice” - maintaining authentic gene expression profiles. It was designed using a mix of Collagenases (Type 4, and A) which provide broad proteolytic activity, but preferentially cleave extracellular bonds, largely leaving cells intact. The total incubation time is 1 hour 20 minutes divided into two layers. At the end of the procedure, RBC lysis is performed. The total yield at the end of the procedure is ~1200 (non-RBC) cells released per mg tissue with 87% viability.</p>
<p> </p>
<p>In the digest mix, there is trypsin inhibitor from soybean which is designed to limit the activity of tryptic proteins in the collagenase mix which can damage the integrity of the cell. There is also 5 mM CaCl2, an activator of collagenase activity, in addition to DNAse - which chews up DNA released from dead cells, reducing cell clumping. The dissociation itself it carried out in two layers. The first layer is 30 minutes and includes trituration and shaking. After this layer, tissue clumps are settled for 1 min, and the supernatant containing released cells is removed and filtered using a 30 µM filter and rinsed with ice-cold PBS-BSA. This helps to preserve the integrity of released cells while continuing the digest clumps of undissociated cells. To the residual clumps, an additional 1 mL of enzyme mix is added and the digestion is continued for 50 additional minutes (1 hr 20 mins total time).</p>
<p> </p>

[GUIDELINES]
<p><strong>Collagenase Enzyme Mix: 2 x 1 mL (make two tubes)</strong></p>
<p>75 µL Collagenase Type A 100 mg/mL (7.5 mg/mL final concentration) </p>
<p>75 µL Collagenase Type 4 100 mg/mL (7.5 mg/mL final)</p>
<p>100 µL of soybean trypsin inhibitor 1 mg/mL (100 µg/mL final)</p>
<p>5 µL DNAse (125 U/mL final)</p>
<p>5 µL of 1 M CaCl2 (5 mM final)</p>
<p>740 µL DPBS (no Ca, no Mg)</p>
<p> </p>
<p> </p>
<p><strong>Reagents</strong></p>
<p>Enzymes, trypsin inhibitor, BSA and DNAse are made up in DPBS (no Ca, no Mg) from Thermo Fisher (14190).<br /> Bovine Serum Albumin - Sigma (A8806).<br /> DNAse - Applichem (A3778) - 10 µL aliquots in PBS each with 250 U.<br /> soybean trypsin inhibitor - Roche (10109886001) - 100 µL aliquots of 1 mg/mL.<br /> Collagenase A - Roche (10103586001) - 100 µL aliquots of 100 mg/mL - frozen at -80 °C.<br /> Collagenase Type 4 - Worthington (LS004186) - 100 µL aliquots of 100 mg/mL - frozen at -80 °C<br /> Red Blood Cell Lysis Buffer - Sigma (R7757)<br /> Trypan Blue Solution 0.4% - Gibco (15250061)</p>
<p> </p>
<p><strong>Equipment</strong></p>
<p>Centrifuge for 1.5 mL, 15 mL conicals<br /> Pipettes and pipet tips<br /> 15, 50 ml Conicals (MLS)<br /> 1.5 mL tubes (MLS)<br /> 30 µM filters - Miltenyi (130-098-458) <br /> Petri dishes (MLS)<br /> Razor blades (MLS)<br /> Ice bucket w/ice<br /> Hemocytometers - InCyto Neubauer Improved (DHC-NO1-5)</p>

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
Protocol for blunting ends by 3' overhang removal and fill-in of 3' recessed (5' overhang) ends using T4 DNA Polymerase.

[GUIDELINES]
CAUTION: Elevated temperatures, excessive amounts of enzyme, failure to supplement with dNTPs or long reaction times will result in recessed ends due to the 3&acute; &rarr; 5&acute; exonuclease activity of the enzyme.&nbsp;<br /><br />* T4 DNA Polymerase can be used in&nbsp;<a href='https://www.neb.com/products/b7201-nebuffer-1-1' target='_blank'>NEBuffers 1.1</a>,&nbsp;<a href='https://www.neb.com/products/b7202-nebuffer-2-1'>2.1</a>, and&nbsp;<a href='https://www.neb.com/products/b7204-cutsmart-buffer' target='_blank'>CutSmart Buffer</a>&nbsp;as well as&nbsp;<a href='https://www.neb.com/products/b7001-nebuffer-1' target='_blank'>NEBuffers 1</a>,&nbsp;<a href='https://www.neb.com/products/b7002-nebuffer-2'>2</a>, and&nbsp;<a href='https://www.neb.com/products/b7004-nebuffer-4' target='_blank'>4</a>&nbsp;and&nbsp;<a href='https://www.neb.com/products/b0202-t4-dna-ligase-reaction-buffer' target='_blank'>T4 DNA Ligase Reaction Buffer</a>. Optimal activity is observed in&nbsp;<a href='https://www.neb.com/products/b7202-nebuffer-2-1' target='_blank'>NEBuffer 2.1</a>. BSA supplementation is recommended when using a buffer that does not already contain BSA.&nbsp;<br /><br />References:<br /><br />1. Tabor, S. and Struhl, K. (1989). DNA-Dependent DNA Polymerases. In F. M. Ausebel, R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith and K. Struhl(Ed.), Current Protocols in Molecular Biology. 3.5.10-3.5.12. New York: John Wiley &amp; Sons, Inc.<br /><br />2. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual. (2nd ed.), 5.44-5.47. Cold Spring Harbor: Cold Spring Harbor Laboratory Press.

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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Since 2012, numerous single-cell RNA sequencing (scRNA-seq) technologies have been introduced. Most involve either significant startup cost, an expert operator, or expensive library preparation. We developed a plate-based library prep that incorporates pre-amplification pooling, unique molecular identifiers (UMIs), and mRNA enrichment using silanol coated magnetic beads. Our method, which builds off of <a href="https://www.nature.com/articles/s41467-017-00136-z" target="_blank" rel="noopener noreferrer">Bush et al. </a>(Nature Communications, 2017) and <a href="https://www.nature.com/articles/nprot.2014.006" target="_blank" rel="noopener noreferrer">Picelli et al.</a> (Nature Protocols, 2014), uses readily available equipment, is easy to automate, and costs a fraction of commercial kits. In addition, our method has been optimized to reduce barcode cross contamination that can occur in pooled pre-amplification reactions. This method allows for plate-based 3’ scRNA-seq with UMIs at relatively low cost, using standard lab equipment.</p>
<p> </p>
<p><span style="text-decoration: underline;">NOTE (2018/08/29)</span>: we are currently testing optimizations described by <a href="https://www.nature.com/articles/s41467-018-05347-6" target="_blank" rel="noopener noreferrer">Bagnoli et al. </a>(<em data-test="journal-title">Nature Communications</em>, 2<span data-test="article-number">018). This protocol may be updated accordingly. </span></p>

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TITLE: MTT assay on 96 well plate
AUTHORS: Adita Ayu Permanasari

[DESCRIPTION]
MTT assay to measure viability cells

[STEPS]
1. Prepare sample in DMSO
2. Observe cell on the plate, make a note about cells condition
3. Experiment by make a serial dilution sample in U-Type, suspend 40 times
4. Remove old medium from cells
5. Put 100 μl/well sample and DMSO, incubate 48 h, 37 oC
6. Observe condition cells after 48 h incubation
7. Checked toxicity under microscope, take photo if there are morphology changing
8. Prepare MTT 10%
9. Remove medium from 96 well plate
10. Put 150 μl/well by multichannel pipet, incubate 4 h, 37 oC
11. Remove MTT solution from well plate
12. Put 100 μl/well DMSO 100% for dissolve
13. Check absorbance at 560 nm and 750 nm , shaker 0.5 min before read absorbance
TITLE: Virus Concentration from Wastewater Using PEG Precipitation and Ultracentrifugation
AUTHORS: Jacquelina.Woods  , rachel.rodriguez  

[DESCRIPTION]
This method was developed at the FDA’s Center for Food Safety and Applied Nutrition for GenomeTrakr’s pandemic response project, monitoring SARS-CoV-2 variants in wastewater​​. Protocols developed for this project cover wastewater collection, concentration, RNA extraction, RT-qPCR detection, library prep, genome sequencing, quality control checks, and data submission to NCBI. This method describes the rapid concentration of intact viruses from wastewater using a combination of PEG precipitation and ultracentrifugation. 

[BEFORE_START]

 
To preserve RNA viruses, samples should be kept on ice whenever possible during the process. To avoid any RNA degradation, sterile glassware and DNase/RNase-free microcentrifuge tubes should be used. 


[STEPS]
SECTION: Virus concentration
1. Place 800 mLof wastewater into an 1L centrifuge bottle.


SECTION: Virus concentration
2. Add 200 µL of extraction control (concentration of 102 per mL), as described here: Preparation of Murine Norovirus for Use as an Extraction Control for Concentration of Viruses from Wastewater (protocols.io). 

SECTION: Virus concentration
3. Add 40 mL of 2X PEG.

 

SECTION: Virus concentration
4. Add 400 µL of 5N HCl.
 

SECTION: Virus concentration
5. Shake briefly and place bottles in refrigerated shaker at   for 120 min.

SECTION: Virus concentration
6. Centrifuge 8000 x g, 30 min, 2-6 °C.


 

SECTION: Virus concentration
7. Carefully pipette supernatant and discard, care should be taken not to disrupt the pellet.
 


SECTION: Virus concentration
8. Resuspend pellet in 15 mL of 0.05M glycine and transfer entire volume to a 50 ml conical tube.
 


SECTION: Virus concentration
9. Incubate on icefor 40 min, vortex occasionally.

SECTION: Virus concentration
10. Add 15 mL of 2X tc PBS to neutralize. Shake by hand to mix.
 

SECTION: Virus concentration
11. Centrifuge at 3000 x g, 15 min, 2-6 °C.

SECTION: Virus concentration
12. Transfer supernatant to clean ultracentrifuge tube (careful not to disturb pellet).
SECTION: Virus concentration
13. Bring total volume up to 65 mL or 125 gtotal weight (includes bottle and cap) with addition of 1X tc PBS.

 

SECTION: Virus concentration
14. Balance tubes to within 0.05 g of each other using 1X tc PBS.

SECTION: Virus concentration
15. Centrifuge at 170000 x g, 45 min, 2-6 °C.

 

SECTION: Virus concentration
16. Discard supernatant and resuspend pellet in 800 µL of 1X tc PBS.
SECTION: Virus concentration
18. Store concentrates from Step 17 at -70 °Cor proceed directly to RNA Extraction from Wastewater Concentrates Using RNeasy and Zymo Kits (protocols.io). 
 


SECTION: Virus concentration
17. Evenly distribute sample into four 2.0 ml microcentrifuge tubes.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
Hybridized Chain Reaction Fluroescent <em>In Situ</em> Hybridization for squid tissue and colonized <em>V. fischeri</em>.

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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Describes novel low conductivity media and multipulse electroporation protocol using a NEPA21 Type II pulse generator with settings optimized to balance transfection efficiency with cell viability for this marine diatom. </p>

[BEFORE_START]
<p>Pseudo nitzschia multiseries cultures are maintained in sterile seawater enriched with Guillard's Marine Enrichment f/2 medium and transferred during mid-log phase.</p>
<p> </p>
<p><span style="text-decoration: underline;">Electroporation Buffer</span></p>
<p> </p>
<p>42% w/v 4K PEG<br />2.5mM Imidazole, pH 7.75</p>
<p> </p>
<p><span style="text-decoration: underline;">Additives*: </span></p>
<p>2mM ATP</p>
<p>2mM MgCl<sub>2</sub></p>
<p>0.5mM Glutathione</p>
<p>0.5mM Ascorbate</p>
<p> </p>
<p>0.2υm filter and store Buffer at 15ºC</p>
<p>*Store additives at -20ºC and add to buffer fresh before each use</p>
<p> </p>
<p><span style="text-decoration: underline;">Note on additions to buffer:</span></p>
<p>We found inclusion of these additives to the electroporation buffer to ease the stress of buffer exchange and electroporation.</p>
<p> </p>
<p><span style="text-decoration: underline;">Additional buffers tried and rejected:</span></p>
<p>Osmolytes: Mannitol, Sorbitol, Tris for pH--&gt; Reduced Pseudo-nitzschia regrowth as compared to PEG</p>
<p>Additives: Proline --&gt; Increased bacterial growth, did not increase viability.</p>
<p> </p>
<p><span style="text-decoration: underline;">Recovery media following electroporation:</span></p>
<p>Sterile seawater ammended with L1 nutrients (Bigelow/ NCMA) at 1:3 NO<sub>3</sub>:Si</p>
<p>Additional: Kanamycin (1ug/mL) was added to the recovery culture following electroporationto in order to limit bacterial growth in culture. The microbiome of Pseudo-nitzschia isolates is apparently coupled to cell growth and physiology (Sison-Mangus, 2014). Growth is impeded following stress and culture takeover by exogenous bacteria.</p>
<p> </p>
<p> </p>

[STEPS]
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TITLE: No Title
AUTHORS: 

[BEFORE_START]
<p>You will need to make the 20 mL of Lysis Buffer prior to running this protocol.</p>

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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol is kit-free and can be used to isolate high quality genomic DNA of predominantly Aiptasia from symbiotic anemones which can be used e.g. as PCR template for genotyping.</p>
<p> </p>
<p>It is based on the method described in Coffroth et al., 1992.</p>

[BEFORE_START]
<p>Recipes:</p>
<p><strong>2×CTAB Buffer</strong></p>
<p><em>(Coffroth et al. (1992) Marine Biology 114: 317-325)</em></p>
<p> </p>
<p><strong>End concentrations:</strong>                                             <strong>For 100 ml buffer mix:</strong></p>
<p>1.4 M              NaCl                                                            28 ml of a 5 M stock solution</p>
<p>20 mM            EDTA (pH 8)                                                 4 ml of a 0.5 M stock solution</p>
<p>100 mM          Tris/HCl (pH 8)                                          10 ml of a 1 M stock solution</p>
<p>2% (w/v)        CTAB powder                                               2 g</p>
<p><u>Add after filter sterilization under a hood:</u></p>
<p>0.2% (v/v)      β-mercaptoethanol (<strong><u>TOXIC!!!</u></strong>)             200 µl</p>
<p> </p>
<ul>
<li>Add ddH<sub>2</sub>O to just under 100 ml.</li>
<li>Warm to 65°C under stirring to bring the CTAB into solution.</li>
<li>Once dissolved, bring final volume to 100 ml using a graduated cylinder.</li>
<li>Filter sterilize (0.2 µm) into sterile 50 ml Falcon tubes and store at −20°C.</li>
</ul>
<p>Heat to 65°C before usage since freezing leads precipitation of the CTAB.</p>
<p> </p>

[GUIDELINES]
<p>Make sure to use Phenol:Chloroform:Isoamyl alcohol (25:24:1) which is buffered with TE to a neutral to slightly basic pH (everthing starting from 6.7 should work) since DNA stability decreases at acidic pH (the opposite is true for RNA).</p>
<p> </p>
<p>Be sure not to exceed the maximal ×g for which your microcentrifuge tubes are certified otherwise they might get damaged during centrifugation and release all the hazards substances (and the DNA of course) into the rotor. You don't want that!</p>
<p> </p>
<p>If making your own stock solutions of RNase A from powder be aware that some products contain DNase impurities. If this should be the case you need to boil the stock solutions once for 15-20 min at 100°C and let cool down at room temperature to inactivate the DNases. </p>

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TITLE: Reverse-phase high pH fractionation (using Thermo Fisher, Cat# 84868)
AUTHORS: Louise Uoselis

[DESCRIPTION]
This protocol uses the Pierce™ High pH Reversed-Phase Peptide Fractionation Kit (Thermo Fisher, Cat# 84868)

[STEPS]
SECTION: Conditioning the columns
1. Remove the white cap on the end of the column and place the column in a 2 mL collection tube.
SECTION: Conditioning the columns
2. Centrifuge at 5000 rcf, 2 min at Room temperature, discard the liquid.

SECTION: Conditioning the columns
3. Remove the screw cap and add 300 µL acetonitrile (ACN) to the column (replacing the screw cap after).

SECTION: Conditioning the columns
4. Centrifuge at 5000 rcf, 2 min at Room temperature, discard the liquid.

SECTION: Conditioning the columns
5. Repeat steps 3 and 4 (for a total of 2 washes with ACN).
SECTION: Conditioning the columns
6. Repeat steps 3 – 5 with 0.1% TFA instead of ACN (total of 2x washes with 0.1% TFA).
SECTION: Conditioning the columns
7. The column is now ready to use.
SECTION: Fractionating the samples
8. Add 300 µL of 0.1% v/v trifluoroacetic acid to each sample.

SECTION: Fractionating the samples
9. Vortex for ~ 10 s

SECTION: Fractionating the samples
10. Leave to incubate for 5 min at Room temperature

SECTION: Fractionating the samples
11. Vortex for ~ 10 s

SECTION: Fractionating the samples
12. Sonicate in a waterbath sonicator for 5 min in an ice slurry.

SECTION: Fractionating the samples
13. Load each sample into a fractionation column, replace the cap and centrifuge at 3000 rcf, 2 min (keep eluate as ‘flow through’ fraction).

SECTION: Fractionating the samples
14. Place the column into a new tube, and load 300 µL of water, and centrifuge at 3000 rcf, 2 min(keep eluate as ‘wash’ fraction).

SECTION: Fractionating the samples
15. Place the column into a new tube, and load the TMT wash solution (5% ACN, 0.1% triethylamine (TEA)) .
SECTION: Fractionating the samples
16. Place the column into a new tube, and load 300 µL of the appropriate elution solution (see table below), and centrifuge at 3000 rcf, 2 minto collect the fraction. 

SECTION: Fractionating the samples
17. Repeat step 5 for each step of the gradient fraction.
SECTION: Fractionating the samples
18. If you are concatenating the fractions, combine the fractions into the desired combinations

SECTION: Fractionating the samples
19. Lyophilise all samples until there are only a few µL left in the tube
TITLE: Total Starch Enzymatic Digestion 
AUTHORS: Lynn   Doran, Amanda P. De Souza

[DESCRIPTION]
Enzymatic digestion of total soluble starch to glucose in plant tissue extracts for preparation for quantification via the GOD-POD Method (NZYtech).

[BEFORE_START]
Extract and dry total starch pellet from plant tissue per Extraction of Non-Structural Carbohydrates (Total Soluble Sugars + Starch) in Plant Tissues.

[STEPS]
1. Prepare fresh daily 120 U/mL α-amylase  (Bacillus licheniformis) in MOPS buffer. 1 mL per sample will be needed. Initial concentration of α-amylase is 3000 U/mL.  Use C1V1 = C2V2 to calculate the volume of α-amylase and MOPS buffer to use.
 

2. Prepare fresh daily 30 U/mL amyloglucosidase (Aspergillus niger) in acetate buffer. 1 mL per sample will be needed. Initial concentration of amyloglucosidase is 3260 U/mL.  Use C1V1 = C2V2 to calculate the volume of amyloglucosidase  and acetate buffer to use.
 

3. Heat the water bath to 75 °C.

4. Add 500 µL of α-amylase in MOPS buffer (120 U/mL) to each sample tube.

5. Vortex to suspend all solids.  Flicking the tube may help dislodge the dried starch pellet.
6. Incubate for 30 min at 75 °C in the water bath.

7. Add another 500 µL of α-amylase in MOPS buffer (120 U/mL) to each sample tube.
8. Vortex to suspend all solids.
9. Incubate for 30 min at 75 °C in the water bath.

10. Cool down the water bath to 50 °C, tubes can be stored at room temp on the counter while water bath cools.
 

11. Add 500 µL amyloglucosidase in acetate buffer (30 U/mL) to each sample tube.

12. Vortex to suspend all solids.
13. Incubate for 30 min at 50 °C in the water bath.

14. Add another 500 µL amyloglucosidase in acetate buffer (30 U/mL) to each sample tube.

15. Vortex to suspend all solids.
16. Incubate for 30 min at 50 °C in the water bath.

17. Place the tubes in ice to stop the reaction, until cool to the touch.
18. Proceed to total starch (as glucose) quantification by NZYtech GOD-POD method or store the samples at -20°C up to one month.
TITLE: Ringer&#39;s Solution
AUTHORS: Mark Ellisman, Mason Mackey, Eric Bushong, Marianne Martone, Thomas Deerinck

[DESCRIPTION]
Standard protocol for preparing 100mL of Ringer's Solution for mice and rat perfusions. 

[GUIDELINES]
Make sure you bubble for at least 5 minutes before adding calcium chloride to Ringer's solution to prevent precipitation. If precipitation occurs, discard and remake entire solution. 

[STEPS]
1. 1.0 mL (Na2HPO4, 18 g/L, at room temperature)
2. 9.9 mL (NaCl, 79.8 g/L, 4 ºC fridge)
3. 1.0 mL (KCl, 37.5 g/L, 4 ºC fridge)
4. 1.0 mL (MgCl2• 6H2O, 20 g/L, 4 ºC fridge)
5. 2.5 mL (NaHCO3, 50 g/L, 4 ºC fridge)
6. 0.200g of Dextrose
8. 1.0 ml Lidocaine hydrochloride
9. 1.0 ml heparin (prevents blood clotting) 
    —bubble 95% air: 5% CO2 at 35 ºC
10. 1.0 mL (CaCl2• 2H2O, 30 g/L, 4 ºC fridge)
7. Bring solution up to 97ml with double distilled water (ddH2O).
TITLE: Mouse perfusion 
AUTHORS: Bryan Killinger

[DESCRIPTION]
General protocol for mouse perfusion fixation.

[STEPS]
1. Anesthetize mouse using mixture of ketamine and xylazine. (Ketamine = 100mg ketamine / kg bodyweight, 10mg xylazine / kg bodyweight)
2. Ensure deep anesthesia with paw pinch. Mouse should not respond.
3. Using four 26g needles, secure mouse paws to corkboard.

4. Make an incision along the abdomen, extending up to the rib cage.
5. Expose chest cavity and secure with hemostats clamped to the xiphoid process.
6. Insert 22g needle into the left ventricle and secure with hemostats.
7. Make small incision in the right ventricle.
8. immediately start peristaltic pump, flowing at a rate of ~8-10mL per min.
9. Once the perfusate runs clear, stop pump, switch to 4% PFA solution, and resume flow.
10. Perfuse mouse with approximately 50-100mL of 4% PFA. 
11. Remove head with scissors, and carefully dissect out the brain.
12. Scull should be snipped at the base, and peeled away carefully. Care should be taken not to damage underlying brain tissues or olfactory bulb.
13. Place dissected brain in 20mL 4% PFA solution, overnight, at 4C.
14. Place in 15% sucrose PBS solution.
15. Once brain sinks, Place in 30% sucrose PBS solution at 4C until sectioning.
16. 0.05% sodium azide can be included in sucrose solutions to prevent bacterial growth.
TITLE: Stereotaxic Injection by Nanoject Protocol
AUTHORS: Allen Institute for Brain Science

[DESCRIPTION]
This protocol describes the delivery of a neuronal tracer using the Nanoject II. The surgery uses a stereotaxic system to target specific brain coordinates in the mouse. 

Note: 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.

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol provides an efficient DNA extraction and purification of ancient sorft tissue. </p>

[BEFORE_START]
<p>Clean</p>

[STEPS]
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