eamag/protocols-with-steps huggingface dataset:
TITLE: Protocol for removing ssDNA from dsDNA or RNA Samples (NEB #M0568)
AUTHORS: New England  Biolabs

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The protocol described below will enable degradation of up to 20 pmol of a 25 nt ssDNA (~ 200 ng). </div><div class = "text-block">In order to degrade larger amounts of ssDNA or ssDNAs longer than 25 nt, we recommend adding more enzyme instead of extending the reaction time. </div><div class = "text-block">Users should note that ssDNAs longer than 25 nt may form secondary structures that hinder Thermolabile Exonuclease I activity.</div></div>

[STEPS]
?. Prepare a  reaction as follows:  AB1Sample containing ssDNA (up to 20 pmol of a 25-mer)x μl2NEBuffer 3.1 (NEB #B7203)*2 μl**3Thermolabile Exonuclease I 1 μl4Nuclease-free water           to 20 μl****Most PCR buffers are compatible.  **No reaction buffer is necessary if enzyme is added to a PCR reaction.***Scale larger reaction volumes proportionally.
20 µl
AB1Sample containing ssDNA (up to 20 pmol of a 25-mer)x μl2NEBuffer 3.1 (NEB #B7203)*2 μl**3Thermolabile Exonuclease I 1 μl4Nuclease-free water           to 20 μl***
?. Incubate at  for .
37 °C
?. Stop reaction by heat-inactivation at  for .
80 °C
TITLE: HuBMAP Lung TMC UCSD &amp; URMC Protocol Overall for Multimodal Single Cell/Nucleus Assays 
AUTHORS: Gloria S Pryhuber, Elizabeth Duong, Blue Lake, Cory Poole, heidie_huyck, Ravi Misra, Xin Sun, Jim Hagood, Dinh H Diep, Kun Zhang

[DESCRIPTION]
This protocol is an overview of the protocols in use for the single nuclear RNA sequencing and ATACseq datasets for HubMAP-Lung Tissue Mapping Center, TMC UCSD / TMC URMC.  It includes protocols.io links to each of the individual protocols that make up this project workflow, from processing tissue, sectioning, H&E imaging the tissue samples, isolation of nuclei from frozen lung tissue, running of multimodal single cell/nucleus assays.uploading the final data to the HuBMAP HIVE.

See also: Human Kidney, Urinary Tract, and Lung Cell Type Mapping  Pipeline for the Human Biomolecular Atlas Program (HuBMAP) V.2   dx.doi.org/10.17504/protocols.io.bvvin64e

[STEPS]
1. Screen and accept donor organs, gross and preserve the tissues into samples for analysis using the protocols: 
 
602.2 Donor Acceptance Criteria for URMC HTC HuBMAP and LungMAP Inclusion  dx.doi.org/10.17504/protocols.io.bjuxknxn
603.3 & 604.5_URMC_HTC_Whole Lung and Lobe Processing
dx.doi.org/10.17504/protocols.io.biz7kf9n
611.2 URMC HTC Formalin-Inflated, Paraffin-Embedded Human Lung Tissue
dx.doi.org/10.17504/protocols.io.bjttknnn
615.1 URMC HTC Non-Inflated Fresh-Frozen Embedded Lung and Associated Tissue
dx.doi.org/10.17504/protocols.io.bjtnknme
615.2 URMC HTC Non-Inflated Fresh-Frozen Embedded Lung and Associated Tissue V2
dx.doi.org/10.17504/protocols.io.rm7vz84k2vx1/v2
3. Register via the HuBMAP UUID generator at https://ingest.hubmapconsortium.org/, the  Donor, the Organ Lobe, the Tissue Block, and the set of tissue sections ( 40 micron curls) from that block used in single nuclear isolation. common coordinate region information. Record and keep locally the key for the generated UUIDs.
4. Register the Tissue Blocks, just registered in the UUID generator, in the Common Coordinate Framework via the Registration User Interface at https://hubmapconsortium.github.io/ccf-ui/rui/
2. Create the cryosections to be used in the single nuclear isolation preparation. Generally placing 10 x 40 micron sections in a single cryovial.
624.1_URMC_HTC_Cryosectioning Non-Inflated Fresh-Frozen Embedded Lung for Nuclear Isolation or Multi-plexed In Situ Hybridization
dx.doi.org/10.17504/protocols.io.3byl4j91jlo5/v1
5. Approximately every 10th section, make a 10 micron section just before and just after, the set of 40 micron curls. Stain these thinner sections by H&E.
621.1.HTC_H&E Stain (Paraffin or Cryosections)  dx.doi.org/10.17504/protocols.io.36wgqjnq3vk5/v1

6. Isolate single nuclei from the tissue curls using protocol:
Frozen lung tissue dissociation for single-nucleus RNA & Chromatin assays 
dx.doi.org/10.17504/protocols.io.bh26j8he
7. Running of multimodal single cell/nucleus assays.
10X snRNA-seq: dx.doi.org/10.17504/protocols.io.86khzcw
10X snATAC-seq: dx.doi.org/10.17504/protocols.io.bvssn6ee
SNARE-Seq2: dx.doi.org/10.17504/protocols.io.bzdsp26e

8. To Be Added: 
10X Chromium Single Cell Multiome ATAC + Gene Expression
MERFISH
DARTFISH
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<h1> </h1>
<h1> </h1>
<h1> </h1>
<p> </p>

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
Protocol to fix marine samples for flow cytometry sorting of phytoplankton. <br />Fix at least 2 samples per depth sampled and at least 6 to 10 depths per vertical profile.<br /><br />Back to the laboratory, pico and nano-plankton populations can be sorted and used for clone library construction or metabarcoding with Next Generation Sequencing (e.g. Illumina).  This was successfully tested on samples from a cruise off Brazil (unpublished data)<br /><br /><strong>Reference<br /></strong>Ribeiro C., Lopes A., Marie D., Vaulot D. Unpublished data<a href="https://www.researchgate.net/publication/265017015_An_Improved_Protocol_for_Flow_Cytometry_Analysis_of_Phytoplankton_Cultures_and_Natural_Samples" target="_blank"><br /></a>

[BEFORE_START]
<strong>Necessary equipment</strong><br /> - Vortex mixer <br />- Cryotubes 2 mL<br />- Liquid nitrogen tank

[STEPS]
?. 
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?. 
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?.
TITLE: Preservation method for long-term storage of fluorescently labeled cells for microscopy
AUTHORS: Jacob Robertson, Erin Garza

[DESCRIPTION]
This method can be used to preserve fluorescently labeled bacterial cells for long-term storage before imaging on a fluorescent microscope.  Using this protocol, samples can be saved at 4°C for weeks-months while maintaining strong, easily detectable fluorescence and cell integrity.

[BEFORE_START]
Prepare the preservation components: 10% glycerol (sterile), 4% paraformaldehyde in PBS pH 7.4 (sterile)

[GUIDELINES]
This protocol has been successfully used on liquid bacterial cultures and on biofilms on solid plastic material.  Cells were imaged using a confocal microscope up to 5 months after preservation.  Fluorescence may be maintained longer than 5 months, but has not been tested by the authors.

[STEPS]
SECTION: Sample
1. Collect your cells (up to 500 μl) in a sterile microfuge tube.
- This protocol can also be used on solid material containing biofilms.

SECTION: Preserve
2. To your sample tube, add 250 µL sterile 10% glycerol and 250 µL sterile 4% paraformaldehyde in PBS (pH 7.4).
- If preserving solid material, make sure the entire sample is submerged in the preservation solution.
SECTION: Preserve
3. Gently Mix
SECTION: Storage
4. Store samples at 4 °C until ready to image.
TITLE: Protocol for ex vivo patch clamping
AUTHORS: Maia Datunashvili

[DESCRIPTION]
Protocol for ex vivo patch clamping

[STEPS]
SECTION: Preparation of acute brain slices
1. Chill sucrose aCSF up to 4 degrees and then keep it on the ice and bubble continuously by carbogen.
SECTION: Preparation of acute brain slices
2. Place 150 ml standard aCSF in holding chamber. Warm it up to 34oC in water bath and bubble continuously by carbogen.
SECTION: Preparation of acute brain slices
3. After 3-5 weeks of the viral injection, anesthetize the animal briefly with Isoflurane and then make IP injection of ketamine/xylazine cocktail (87/13 mg/kg i.p.).
SECTION: Preparation of acute brain slices
4. Perfuse the animal transcardially with 4-6 ml ice-cold sucrose aCSF.
SECTION: Preparation of acute brain slices
5. Open the scalp, block the brain between the hemispheres and transfer it to the petri dish filled with ice-cold oxygenated sucrose solution.
SECTION: Preparation of acute brain slices
6. Glue the brain on the specimen plate and cut 250 um thickness slices on the vibratome.
SECTION: Preparation of acute brain slices
7. Wash the slices with standard aCSF and transfer them to the holding chamber. Keep the slices 30 min at 340C and then at room temperature.
TITLE: QMask Hemispheric Separation
AUTHORS: Michael X. Henderson

[DESCRIPTION]
This protocol describes QMask hemispheric separation.

[STEPS]
SECTION: QMask Hemispheric Separation
1. Open the Qmask tool and click “pick XML” and open the QuickN XML file generated in QuickNII.
SECTION: QMask Hemispheric Separation
2.  Enter the appropriate coordinates from the QMask Coordinates file.
SECTION: QMask Hemispheric Separation
3. Click “Destination” and navigate to the QVN\Mask folder. Click “Go”.
 The mask output should be a black and White PNG that looks something like this:
 
SECTION: QMask Hemispheric Separation
4.  Rename the mask files using the appropriate naming convention. Example: NG18I_s061_mask.
TITLE: Formation of gp37 LTF needle trimers-OmpC complex
AUTHORS: Mohammad Z. Islam, Andrei Fokine, Marthandan Mahalingam, Zhihong Zhang, Carmela Garcia-Doval, Mark J. van Raaij, Michael G. Rossmann, and Venigalla B. Rao

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Tailed bacteriophages (phages) are one of the most abundant life forms on Earth. They encode highly efficient molecular machines to infect bacteria, but the initial interactions between a phage and a bacterium that then lead to irreversible virus attachment and infection are poorly understood. This information is critically needed to engineer machines with novel host specificities in order to combat antibiotic resistance, a major threat to global health today. The tailed phage T4 encodes a specialized device for this purpose, the long tail fiber (LTF), which allows the virus to move on the bacterial surface and find a suitable site for infection. Consequently, the infection efficiency of phage T4 is one of the highest, reaching the theoretical value of 1. Although the atomic structure of the tip of the LTF has been determined, its functional architecture and how interactions with two structurally very different Escherichia coli receptor molecules, lipopolysaccharide (LPS) and outer membrane protein C (OmpC), contribute to virus movement remained unknown. Here, by developing direct receptor binding assays, extensive mutational and biochemical analyses, and structural modeling, we discovered that the ball-shaped tip of the LTF, a trimer of gene product 37, consists of three sets of symmetrically alternating binding sites for LPS and/or OmpC. Our studies implicate reversible and dynamic interactions between these sites and the receptors. We speculate that the LTF might function as a “molecular pivot” allowing the virus to “walk” on the bacterium by adjusting the angle or position of interaction of the six LTFs attached to the six-fold symmetric baseplate.</div></div>

[STEPS]
TITLE: Transfection of Diplonemids using AMAXA Nucleofactor apparatus
AUTHORS: Binnypreet Kaur1, 2 ,  Drahomíra Faktorová1, 2,  Priscila Peña-Diaz1 and Julius Lukeš1, 2

[STEPS]
?. [Program: X-001/ X-014]
Count the cells and plan to 1 nucleofection with 5x 107 cells for each construct.
?. [Program: X-001/ X-014]
Harvest the cells by centrifugation at 1300xg for 5 min at room temperature in Swing Bucket Rotor
?. [Program: X-001/ X-014]
Resuspend the cell pellet in 100ul of AMAXA Human T- cell solution at 4°C (from refrigerator combine 81.8ul of Human T-cell nucleofector solution + 18.2ul Supplement).
?. Add 5-10ug of (PCR) (linearized DNA) into cuvette(resuspend in 10ul of H2O). Do not add anything in case of Negative control
?. Put everything into the cuvette, close the cap and place in the electroporator, cuvette should only fit in one direction, but metal sides should face towards you.
?. Press for the Program X-001/X-014 to electroporate.
?. Transfer the entire cell- DNA transfectant into 10ml of Diplonema media (chloramphenicol) with the supplied disposable micropipettes.Note: Make sure labeled flasks with media should be ready before electroporation.
?. Immediately observe the flasks containing transfectants under a microscope and place them in the incubator at 27C + shaker and let them grow for 6-8h
?. After 6-8 hours took out the flasks and make controls which should be without antibiotics  - put 1ml of the electroporated cells in the first well of 24 well plates
?. In the rest - 9ml -transfectants add antibiotics (HYG or G418or Puro) on the basis of the selection marker in the flasks
?. Add 1.5ml of electroporated cells in the first row  (6 wells) of 24 well plates. Put 900ul of the media (+ selectable drug) in the rest of the wells. Take 100ul from first well and transfer to the next to make 10x serial dilution and so on...up to the lowest concentration 5 x 102 cells/ml.
?. Do the same  with the negative control plating.
TITLE: Microsomal membrane isolation from cell culture
AUTHORS: rosanne.wouters, Peter Vangheluwe

[DESCRIPTION]
Microsomal membrane isolation from cell culture

[STEPS]
SECTION: harvest cells
1. harvest cells by detachment with TrypLE Express (Gibco, 12604-021)

SECTION: harvest cells
2. pellet cells by centrifugation (5 min, 400 gavg, 4°C) 
SECTION: harvest cells
3. wash cell pellet in phosphate buffered saline (PBS, Sigma, D8537-500ml)
SECTION: harvest cells
4. pellet cells by centrifugation (5 min, 400 gavg, 4°C) 
SECTION: lyse cells
5. resuspend pellet in RIPA lysis buffer (Thermo Scientific, 89900) supplemented with protease inhibitor (Sigmafast, Sigma, S8830-20TAB) 
SECTION: lyse cells
6. incubate 30min on ice
SECTION: lyse cells
7. centrifuge lysate (10min, 100 gavg, 4°C) to pellet and remove nuclear fraction
SECTION: isolate microsomal fraction
9. centrifuge supernatant from previous step (10min, 15 000 gavg, 4°C) to pellet the mitochondrial/lysosomal fraction
SECTION: lyse cells
8. collect supernatant to continue isolation
SECTION: isolate microsomal fraction
10. collect supernatant to continue isolation (pelleted mitochondrial/lysosomal fraction can be stored for future analysis)
SECTION: isolate microsomal fraction
11. centrifuge supernatant from previous step (35 min, 200 000 gavg, 4°C) to pellet the microsomal fraction
SECTION: isolate microsomal fraction
12. resuspend pellet in sucrose (0.25 M)
SECTION: isolate microsomal fraction
13. for Western blotting prepare samples with 20µg protein with 4x LDS loading buffer (NuPAGE, invitrogen, 2399420)
TITLE: Virus purification protocol for Ehv-163 and other viruses.
AUTHORS: Elena Yakubovskaya, Tatiana Zaliznyak, Joaquin Martinez Martinez, Gordon Taylor

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Reliable protocol for virus purification is an essential step for characterization and identification of individual virus particles in order to obtain information on biochemical and physical properties of the virus and for studying interactions between host and virus at the single-cell level. The purified virus should be physically and chemically undamaged by the purification procedure and free from contaminating host-material. To obtain high-quality EhV sample (the virus that infecta coccolithophorid,</span><span style = "font-style:italic;"> Emiliania huxleyi) </span><span>sutable for AFM, Raman and CryoEM analysis,</span><span style = "font-style:italic;"> </span><span> we modified the protocol published by Lawrence and Steward (</span><span style = "font-style:italic;">“Purification of viruses by centrifugation” J. E. Lawrence and G. F. Steward; MAVE Chapter 17, 2010, 166–181 © 2010, by the American Society of Limnology and Oceanography, Inc.). </span><span></span></div></div>

[STEPS]
?. Concentrate lysate 50 times by tangential flow filtration with a Vivaflow 50 cartridge (100 KDa MWCO) to decrease volume. For EhV-163 virus, the final volume was 50 ml and the final concentration was  ~7 x 109EhV/ml.
?. Prepare OptiPrep solutions using F/2-Si media or 150mM NaCl, 20mM HEPES pH 8.0 buffer. 1mM EDTA buffer as the diluent. For many viruses, a gradient from 20%- 50% OptiPrep will provide a good range for separation, but very dense or light viruses may require adjustment. For EhV-163 virus, we used 20%, 25%, 30%, 35%, 40% and 50% v/v OptiPrep solutions (OptiPrep is sold as a 60% solution).
?. Using the underlying technique with a syringe and pipetting needle, pour 6-step gradients into open-toped ultracentrifuge tubes, beginning with the least dense solution first. 1ml 20%, 2ml 25%, 2ml 30%, 2ml 35%, 2ml 40%, 1ml 50%.Allow blending for 2 h at room temperature.
?. Carefully overlay the gradient with virus concentrate. For the 10-mL gradient, we used 2-mL concentrated virus. For SW-41 ultracentrifuge rotor 4h at 26,000 rpm (100,000g) is sufficient h for good separation of Ehv-163 but overnight run also works. Allow the rotor to decelerate using a slow deceleration program below 4000 rpm or turn off the brake below 4000 rpm.
?. Virus bands are visible with a regular flashlight. For harvesting a single band in a density gradient use side or bottom puncture with a needle.
?. To remove iodixanol from the sample of Ehv-163  use 3x dialysis against 1L of 150mM NaCl, 20mM HEPES pH 8.0, 1mM EDTA buffer using Float-A-Lyzer G2 with 100kD cut off dialysis membrane.
?. Equilibrate 3 ml of CaptoCore 700 resin with  10ml of 150mM NaCl, 20mM HEPES pH 8.0, 1mM EDTA buffer.  Purify  EhV-163 on a Capto Core 700 resin using in batch or column method to remove traces of iodixanol. Aliquot purified virus sample Store virus sample in al at 4°C in the dark.
TITLE: Immunofluorescence for adherent cells
AUTHORS: Alexandros C Kokotos, Tim Ryan

[DESCRIPTION]
This protocol describes a method to perform immunostaining for adherent cells.

[BEFORE_START]
This protocol describes a method to perform immunofluorescence on adherent cells on coverslips. It can also be used for immunocytochemistry by changing the secondary antibodies and also adjusted for cells growing on plastic. This protocol has been verified for use with both cell line cells and primary cells, like neurons.

[GUIDELINES]
Cells should be cultured and handled following all local biosafety regulations.

[STEPS]
SECTION: Immunofluorescence
1. Fix cells with 4 % (v/v) PFA in PBS for 10 min.
Note: Discard PFA in a special container and not in the drain.
SECTION: Immunofluorescence
2. Quench PFA by using a 50 millimolar (mM) NH4Cl solution in PBS for 10 min.

SECTION: Immunofluorescence
3. Permeabilize cells with 0.25 % (v/v) Triton X-100 in PBS for 10 min.

SECTION: Immunofluorescence
4. Block with 4 % (v/v) BSA in PBS for 60 min.
Note: At this step the protocol can be paused. Blocking can also be done overnight.
Other blocking solutions can be used, such as goat or donkey serum matching with the species of the secondary antibodies used.
SECTION: Immunofluorescence
5. Incubate primary antibodies in 4 % (v/v) BSA in PBS for 60 min.
For adherent cells on glass coverslips, the antibody solution is drop seeded on parafilm and the coverslip is placed upside down on the antibody solution using the inverted tweezers. For the 22x22 mm coverslips, 100 ul of antibody solution is sufficient for the whole coverslip to be incubated with antibody. For cells growing on plastic the antibody solution can be added in the well/dish.
SECTION: Immunofluorescence
6. Wash cells with PBS for 5 min three times. 

SECTION: Immunofluorescence
7. Incubate secondary antibodies in 4 % (v/v) BSA in PBS for 60 min.
For immunofluorescence, we usually use Alexa dye secondary antibodies (Thermo Scientific) at a 1:500 dilution.
SECTION: Immunofluorescence
8. Wash cells with PBS for 5 min three times. 
SECTION: Immunofluorescence
9. Wash cells with ddH2O to remove excess salt and protein crystals.
For cells on coverslips, the coverslips are dipped into a beaker with ddH2O four times and excess ddH2O is removed by touching the edge of the coverslips on absorbent paper.
SECTION: Immunofluorescence
10. Add the coverslip on a glass slide that has been previously drop seeded with Prolong Diamond mountant. Place coverslips inverted and avoid trapping any air in the process. Allow to air dry overnight in a dark environment. 
For the 22x22 mm coverslips, 50 ul of mountant is sufficient. Pipette mountant with a cut tip to avoid bubbles.
SECTION: Immunofluorescence
11. Seal the coverslips the next day using clear nail polish.
TITLE: Yale - Alkaline Phosphatase
AUTHORS: Gary Cline, John Stack

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary: </span></div><div class = "text-block">Procedure to measure the amount of Alkaline Phosphatase activity. Alkaline Phosphatase (ALP) activity is measured from the hydrolysis of 4-nitrophenylphospate to 4-nitrophenyoxide ion (monitored at 405 nm) and phosphate. </div></div>

[STEPS]
?. Calibrate Cobas for Alkaline Phosphatase Activity analysis by running two assayed control serum.
?. Sample handling as performed by the Cobas Mira Plus.    a) Pipette 3 µL of sample into a cuvette slot.    b) Add 150 µL of Alkaline Phosphatase Reagent.    c) Mixture is incubated at 37˚C and spun for 10 minutes.    d) Absorbance is measured at 405 nm.
TITLE: Study Vaccine (Part 5 of Phase 3 study of Vaccine Candidate for COVID-19)
AUTHORS: Chris Ockenhouse, Chris Gast, Renee Holt, Jorge Flores

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This is Part 5 of "Phase 3 randomized, double-blinded, placebo-controlled trial to evaluate the safety, immunogenicity, and efficacy of </span><span style = "font-weight:bold;">Vaccine Candidate</span><span> against COVID-19 in adults > 18 years of age"</span></div><div class = "text-block">This generic Phase 3 protocol was developed by the PATH team with support of the Bill and Melinda Gates Foundation. The aim of the collection is to share recommended best practices in designing and implementing a Phase 3 study of a COVID-19 vaccine candidate. As Phase 3 trials of different Vaccine Candidates  proceed around the world, following the same protocols will ensure consistency and comparability of the Phase 3 trial results.</div><div class = "text-block"><span style = "font-weight:bold;">Please note</span><span> that this is an evolving document, to be versioned and updated, based on community feedback and new data.</span></div></div>

[STEPS]
TITLE: pAmpPSBA Plasmid Map
AUTHORS: Isabel Nimmo, Ellen Nisbet, Adrian Barbrook, Christopher Howe

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>A plasmid map for the</span><span style = "font-style:italic;"> Amphidinium carterae </span><span>pAmpPSBA construct</span></div></div>

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
A real-time assay for the detection of Rubella virus RNA from clinical samples. This assay was modified from a published method with oligonucleotides updated, a different kit used, and cycling times adjusted accordingly.


[GUIDELINES]
<ul>
<li>If using a different brand or model of real-time thermocycler, check the concentration of ROX is adequate.</li>
<li>Method assumes the user is familiar with the thermocycler and software used to run the protocol.</li>
</ul>

[STEPS]
SECTION: Oligonucleotide sequences
?. 
SECTION: Reaction set-up
?. 
SECTION: Amplification
?. 
SECTION: Result Analysis
?.