eamag/protocols-with-steps huggingface dataset:
TITLE: 10x Protocols: Chromium Single Cell/Nuclei Gene Expression Flex Fixation -- University of Minnesota  TMCs (CG000478 Rev B)
AUTHORS: IOx Genomics, Laura J Niedernhofer, David A Bernlohr

[DESCRIPTION]
DOIs for dissociation protocols and 10x Genomics fixation for Chromium Single Cell Expression flex protocols. 

Please see DOIs for dissociation protocols linked here.

Protocol ID# (CG) and Revision letter provided here: 
    10x CG000478, Revision B -- Fixation of single cells and nuclei prior to applying 10x Genomics Chromium flex protocols. 

Note: These protocols may not be the current version offered by the company but were used to produce the specific datasets connected to them. Please review the company support websites for the most recent versions of the protocols prior to starting your experiment. 

[STEPS]
1. https://www.10xgenomics.com/products/single-cell-gene-expression-flex
https://www.10xgenomics.com/support/single-cell-gene-expression-flex
2. 10x Protocol CG000478, Rev B (Fixation):
TITLE: UC Davis - Luminex/Multiplex
AUTHORS: Lori Haapanen

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary: </span></div><div class = "text-block">This core will provide services for quantification and comparison in animal models for hormone measurements; kidney function analytes; lipid metabolism analytes; liver function analytes; miscellaneous protein analytes; and immune function including cytokines/chemokines. The Luminex assay enables the quantification of all or any combination of analytes for which there are assay beads in tissue/cell lysate, tissue culture supernatant samples and plasma/serum. For example, cytokines and chemokines are peptides, which act as regulators in normal and pathological conditions, effecting interactions between cells as well as regulating processes occurring in the extracellular environment.</div></div>

[STEPS]
?. Pre-wet the filter plate with 200 ul assay buffer, agitate, incubate for 10 minutes RT and vacuum.
?. Add 25 ul of standards, controls, assay buffer, samples, matrix serum and beads to appropriate wells on the filter plate.
?. Place filter plate on plate stand, seal, wrap in foil and incubate overnight while agitating on a Titer Plate Shaker at 4 degrees C (16-18 hours).
?. Remove fluid from the filter plate by vacuum and wash. Bolt well.
?. Add 25 ul of detection antibody to each well, place filter plate on plate stand, seal, wrap in foil, agitate on a Titer Plate Shaker for 1 hour at RT.
?. Without removing detection antibody, add 25 ul of Streptavidin- Phycoerythrin to each well.  Place filter plate on plate stand, seal, wrap in foil, agitate on the Titer Plate Shaker.  Incubate for 30 mins RT.
?. Remove the fluid by vacuum and wash the plate. Bolt well.
?. Add 150 uls of Sheath Fluid to each well, place on plate stand, seal, wrap in foil, agitate on the Titer Plate Shaker at RT for 5 minutes.
?. Set up protocol on the Bio-Plex 200: add analytes and regions, standards, quality controls and sample dilutions to Bio-Plex 6.0 Manager software.
?. Run plate on Bio-Plex (Luminex) 200.
?. Save and analyze the median fluorescent intensity data using a 5-parameter logistic or spline curve fitting method for calculating cytokine/chemokines concentrations in samples in pg/ml.
TITLE: PROTEIN INJECTION PROTOCOL
AUTHORS: Scott Vermilyea

[DESCRIPTION]
This protocol details the injection parameters of the protein.

[STEPS]
SECTION: Injection Parameters
1. Stereotaxic coordinates:
SECTION: Injection Parameters
1.1. Intracortical/Intrastriatal (IC/IS): 2.0 mm lateral from the midline, +0.2 mm relative to bregma, and 0.8 and 2.6 mm deep from the dura.
SECTION: Injection Parameters
1.2. Brainstem: 0.2 mm lateral from the midline, -7.34 mm from bregma, 3.75 mm deep from the dura.

SECTION: Injection Parameters
2. Injectable:
SECTION: Injection Parameters
2.1. 2.5 µg of total protein in a volume of 2.5 µL. 

SECTION: Injection Parameters
2.2. Rate: 0.1 µL per minute

SECTION: Injection Parameters
2.3. Needle: 28 ga needle attached via tubing to a Hamilton syringe controlled by a constant pressure syringe pump (Harvard Apparatus, Holliston, MA).
TITLE: Preparing single-cell suspension from human embryo/fetus
AUTHORS: Sten Linnarsson, Emelie Braun

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Protocol for human embryo/fetal tissue handling and dissociation, suitable for subsequent single-cell RNA sequencing and in situ hybridization.</div></div>

[STEPS]
?. [Preparations]
Oxygenate Earl's Balanced Salt Solution (EBSS) by bubbling with 95% O2:5% CO2 on ice for 5-10 min (until appropriate pH is reached).
?. [Preparations]
Add 5 mL EBSS to Papain vial, dissolve at , then move to room temperature (RT).
For embryonic/foetal heart dissociation, 5 mL of collagenase II is used with a concentration of 200 U/mL instead of Papain.
?. [Preparations]
Start pre-cooling the centrifuge at 4-8 °C while dissolving the enzymes. This is a good time to take images of the tissue and prepare the dissection instruments used for cutting the tissue.
?. [Dissociation]
Cut the tissue into smaller pieces and transfer the pieces to the Papain vial using a pair of tweezers.
?. [Dissociation]
Incubate the tissue for 25-40 min at . Incubation time may vary depending on age and region. Younger tissue usually requires shorter time.
For embyronic/foetal heart dissociation, the incubation time varies between 40 min - 1 h or more.
?. [Dissociation]
After incubation, carefully triturate the tissue until pieces fall apart by using a fire polished Pasteur pipette.If undissociated pieces are still present, continue incubation 2-5 min more and triturate again (pieces should fall apart easily).
?. [Dissociation]
Filter the dissociated cells through an EBSS-equilibrated cell strainer of Partec filter.Spin the cells at 200 g for 5 min at 4-8 °C. (If needed, perform gradient centrifugation: resuspend the pellet in 900 μL EBSS, 100 μL Albumin inhibitor, 50 μL DNase and carefully layer on top of 3 mL Albumin inhibitor solution. Centrifuge at 70 g for 6 min at 4-8 °C with slow acceleration and deceleration). This step can be used to remove blood cells from cell suspensions of the heart.  Spin the cells at 200 g for 5 min at 4-8 °C. (If needed, perform gradient centrifugation: resuspend the pellet in 900 μL EBSS, 100 μL Albumin inhibitor, 50 μL DNase and carefully layer on top of 3 mL Albumin inhibitor solution. Centrifuge at 70 g for 6 min at 4-8 °C with slow acceleration and deceleration). This step can be used to remove blood cells from cell suspensions of the heart.
?. [Dissociation]
Remove the supernatant and resuspend the pellet in approximately 100 μL (or more if too concentrated) with a fire polished Pasteur pipette. Transfer the cell suspension to a 1.5 mL Eppendorf tube pre-coated with 30% bovine serum albumin (BSA). Count the cells using a hemocytometer or an automated cell counter. Adjust the concentration as desired for the 10X Chromium platform. Transfer the cell suspension to a 1.5 mL Eppendorf tube pre-coated with 30% bovine serum albumin (BSA). Count the cells using a hemocytometer or an automated cell counter. Adjust the concentration as desired for the 10X Chromium platform.
TITLE: Bogus protocols: test access to process &amp; material info
AUTHORS: Abby   Moore

[DESCRIPTION]
The purpose of this protocol is to test accessing the material and process components and icons.

[STEPS]
SECTION: This is the first section
1. In this step, I'll use the centrifugation component and icon.

37000 rcf, 2 min, -20 °C 

SECTION: This is the first section
2. In this step, I'll use the shaker component.

  

SECTION: This is the first section
2.1. In this substep, I'll use the incubation icon.
SECTION: This is the first section
3. In this step, I'll use the pipetting icon.
SECTION: This is the first section
3.1. In this substep, I'll use the wash icon.
SECTION: This is the first section
3.2. In this substep, I'll use the mix icon.
TITLE: Learn Partial Correlation Disease-Specific Networks
AUTHORS: Lillian Thistlethwaite

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes how to construct disease-specific network structures as described in Thistlethwaite et al. (2020). </div><div class = "text-block">Thistlethwaite L.R., Petrosyan V., Li X., Miller M.J., Elsea S.H., Milosavljevic A. (2020). CTD: an information-theoretic method to interpret multivariate perturbations in the context of graphical models with applications in metabolomics and transcriptomics. In review.</div></div>

[STEPS]
?. [Prepare dataset]
?. [Prepare dataset]
Learn disease-specific network folds for three different network learning paradigms for 5 disease states (citrullinemia, maple syrup urine disease, methylmalonic aciduria, propionic aciduria, phenylketonuria):i)   latent embedding + network pruning ("ind")ii)  latent embedding + no network pruning ("noPruning")iii) no latent embedding or network pruning ("noLatent")"ind" networks will be the ig_pruned R objects saved in .RData files in a folder called ind_foldNets."noLatent networks will be the ig R object saved in .RData files in a folder called noLatent_foldNets."noPruning" networks will be the ig R object in the ind_foldNets .RData files.Each disease-specific network takes an average of 3-5 minutes to learn. We learn a total of CIT(9)*2+MSUD(18)*2+MMA(9)*2+PA(9)*2+PKU(8)*2 = 106 network folds, 53 per network learning paradigm ("ind" versus "noLatent"). In total, all networks were learned within 5-9 hours.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
For use in <a href="https://www.protocols.io/view/Polyacrylamide-Gel-System-For-Electrophoresis-Of-P-ep7bdrn" target="_blank">Polyacrylamide Gel System For Electrophoresis Of Proteins</a>.

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

[DESCRIPTION]
<p>This protocol provides a procedure to generate taxonomic data from assembled contigs using centrifuge. </p>

[GUIDELINES]
<p><a href="http://www.ccb.jhu.edu/software/centrifuge/manual.shtml" target="_blank">Centrifuge documentation</a></p>

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

[DESCRIPTION]
Isobaric peptide termini labeling (IPTL) is a quantification method which permits relative quantification using quantification points distributed throughout the whole tandem mass spectrometry (MS/MS) spectrum. It is based on the complementary derivatization of peptide termini with different isotopes resulting in isobaric peptides.

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

[DESCRIPTION]
For <a href="https://www.protocols.io/view/Transcriptomics-During-One-Step-Growth-Curves-for-dem3c5" target="_blank">Transcriptomics During One-Step Growth Curves for Cellulophaga Phages protocol</a>.

[STEPS]
?. 
?. 
?. 
?.
TITLE: Vector Digestion and Purification
AUTHORS: Carolina Lopez

[DESCRIPTION]
Protocol for plasmid digestion and purification

[STEPS]
SECTION: Isolate Digested Vector:
2. 1. Digest Vector with New England Biolabs Restriction Enzymes:
 
2. Incubate for 120 min at 37 °C.
3. Add 10 µL of 6x loading buffer to reaction and vortex briefly to mix.
4. Make 1% low melt-agarose gel.
        a) Mix 1 g of Agar with 100 mL of TAE Buffer.
        b) Microwave to boil agarose and let cool until you can touch bottle, but gel is not solid.
        c) Add 1.5 µL of EtBr to agarose and pour into DNA gel mold with 10 well comb.
        d) Let gel solidify.
5. Load 60 µL of reaction into well of gel
6. Run gel for 45 min at 120V.
7. Visualize band with UV light and cut out section of gel with band with new razor blade and place in 1.5 mLtube. 
8. Purify Band from gel with Promega Wizard SV Gel and PCR Purification Kit (A9282)
        a) https://www.promega.com/products/nucleic-acid-extraction/clean-up-and-concentration/wizard-sv-gel-and-pcr-clean-up-system/?catNum=A9281
        b) Weigh DNA gel fragment and add 10 µL of Membrane Binding Solution per 10 mg of gel slice. 
        c) Incubate mixture at 65 °C for 10 min or until gel is completely melted.
        d) Add melted gel mixture to SV minicolumn in Collection Tube and incubate at room temperature for 1 min.
        e) Centrifuge at max speed for 1 min. Discard flowthrough and reinsert column into tube.
        f) Add 700 µL of Membrane Wash Solution. Centrifuge at max speed for 1 min. Discard flowthrough and reinsert column into tube.
        g) Add 500 µL of membrane wash solution. Centrifuge at max speed for 1 min. Discard flowthrough and reinsert column into tube.
        h) Spin empty column for 1 min at max speed to remove excess ethanol.
        i) Transfer column to labelled 1.5 mL tube and add 35 µL of NF H20. Incubate for 1 min at room temperature. 
        j) Centrifuge at max speed for 1 min. Keep eluate and store at -20 °C.

SECTION: Isolate Digested Vector:
1. Gibson Assembly technology uses homologous recombination to assemble adjacent DNA fragments that share end-terminal homology. The optimal length of the homologous fragment ends region depends on 
the number and length of the fragments in the assembly reaction.
TITLE: OSU TriState SenNet Normal Donor Lung Acceptance Criteria
AUTHORS: Sean D. Stacey, Brenda F. Reader, Lorena Rosas, Victor Peters, Ana L. Mora, mauricio.rojas

[DESCRIPTION]
This protocol describes the criteria required for inclusion of human lung specimen from healthy “normal” organ donors in the Comprehensive Transplant Center Human Tissue Biorepository and TriState SenNet Biospecimen Core at The Ohio State
University as part of the Cellular Senescence Network Program (SenNet). This protocol is based on the CTC Human Tissue Biorepository's standard operating procedure established for tissue collection for explant and donor samples. We follow
routine laboratory specimen processing guidelines to ensure the highest biospecimen quality.

[STEPS]
SECTION: Inclusion Criteria
1. Donor is consented for research by Lifeline of Ohio or registered organ donor, 18-80 years of age, negative for HCV, HBV, HIV, and no history of latent tuberculosis or active COVID-19
infection. Depending on the investigators’ request we remove specimens from upper, lower, or both lobes of a single lung.
When processing “normal” donor lung tissue, we choose samples from the healthiest section of the lung. We refer to the CT scan or chest x-ray to locate these regions.

For “normal” donor lungs, we look for the following characteristics:

1. Tissue is spongy.
2. Tissue is pink in color and color is uniform.
3. Absence of abscesses, nodules, or emphysematous presentations. 
SECTION: Exclusion Criteria
2. Donor is 17 years of age or younger, HBV, HCV, or HIV positive, has a latent
tuberculosis infection or active COVID-19 infection.
TITLE: UC Davis - Electrocardiography
AUTHORS: Anil Singapuri

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary: </span></div><div class = "text-block">Cardiac hypertrophy is one of the most common causes of heart failure. The development of cardiac hypertrophy and failure can be monitored using electrocardiography (ECG) in conscious animals. ECG is used to measure the rate and regularity of heartbeats as well as the size and position of the chambers, the presence of any damage to the heart, and the effects of drugs or devices used to regulate the heart.</div></div>

[STEPS]
?. Mice will be anesthetized with intraperitoneal ketamine 100 mg/kg and xylazine 5 mg/kg, intubated and mechanically ventilated with supplemental oxygen at a respiratory rate of 100 breaths/min and tidal volume of 0.2 ml.
?. The fur will be removed from the surgical incision sites (Nair) and the skin cleaned with Betadine followed by 70% alcohol 3 times.
?. Surgery will be performed on a clean and disinfected lab bench, with sterilized instruments (autoclaved). Mice will be on a sterilized cloth on a circulating water blanket to maintain body temperature. Surgeons will scrub with Betadine, mask and wear sterile gloves.
?. A 10-mm incision will be made in the abdomen and the sterile transmitter placed within the peritoneal cavity and will be secured to the inside wall of the abdomen with non-absorbable sutures.      a. ECG transmitter (Data Sciences International, volume=2.1 cc and weight=3.4 g)
?. The abdominal muscle will be closed with 6-0 Nylon sterile sutures and the skin will be closed with 4-0 absorbable sutures (Ethilon).
?. A bolus injection of 0.1 mg/kg of buprenorphine subcutaneous will be given.
?. Continuous ECG signals can be recorded in freely moving mice in their home cage at various time periods.
?. The two ECG leads will be tunneled subcutaneously and tied to right shoulder and xyphoid space caudal to the ribcage.
TITLE: Assay for the Enzymatic Degradation of PET Beads
AUTHORS: Joao Vitor Molino

[DESCRIPTION]
This assay focuses on assessing the enzymatic degradation of PET beads through absorbance measurements at 240nm. The primary goal is to quantify the enzymatic activity on PET beads by monitoring the release of BHET. This is achieved using a UV-transparent microplate, with absorbance readings specifically taken at 240nm.

[GUIDELINES]
Ensure the supernatant transfer is consistent in volume across all samples.

The enzyme and PET bead concentrations might need optimization based on the sensitivity of the assay and the activity of the enzyme.

A negative control with an inactivated enzyme can help determine the baseline of non-enzymatic degradation.

Adjustments to the protocol may be necessary based on specific experimental parameters, enzyme characteristics, or the sensitivity required for the assay.

[STEPS]
SECTION: Reaction Setup
1. In each PCR tube, add 30mg of PET plastic beads.
SECTION: Reaction Setup
2. Add 100 µL of 1 Molarity (M) potassium phosphate buffer to the tubes

SECTION: Reaction Setup
3. Add 100 µL of the enzyme solution to the tubes with PET beads to initiate the reaction. These constitute the test samples.

SECTION: Reaction Setup
4. Prepare a blank control by adding 100 µL of buffer and 100 µL of enzyme solution into a PCR tube without PET beads.

SECTION: Incubation
5. Place the PCR tubes in the thermocycler.
SECTION: Incubation
6. Set the thermocycler to 68 °C with the lid heated to 105 °C to prevent condensation. Incubate for 12 hours.

SECTION: Sampling
7. Following the incubation period, briefly cool the PCR tubes to room temperature.
SECTION: Sampling
8. Centrifuge the tubes if necessary to ensure that the PET beads settle to the bottom.
SECTION: Sampling
9. Carefully transfer 100 µL of the supernatant from each tube into the corresponding wells of the UV-Star™ 96-well Microplate.

SECTION: Absorbance measurement
10. Measure the absorbance of each well at 240 nm using the TECAN plate reader. Ensure to measure both the test samples and the blank control.
SECTION: Data Analysis
11. Calculate the enzymatic activity by comparing the absorbance readings of the test samples with that of the blank control. The difference in absorbance is indicative of BHET release and, consequently, of PET degradation.
TITLE: RT-PCR multiplex en Tiempo Real para la detección de virus SARS-CoV-2
AUTHORS: Priscila Nayu Lope Pari, Maribel Huaringa Nunes, Carlos Padilla Rojas, Nancy Rojas Serrano, Janett Protilla Romero, Gloria Arotinco Garayar, Lely Solari Serpa

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Validar el método de ensayo del  “RT-PCR multiplex en Tiempo Real para la detección del virus SARS-CoV-2”  a partir de muestras clínicas respiratorias para la detección </span><span style = "font-style:italic;">in vitro</span><span> del virus  SARS-CoV-2 mediante los parámetros establecidos para este método aplicado en el laboratorio de Virus Respiratorios.</span></div><div class = "text-block">Se realizó la validación debido a que este método está basado en protocolos publicados en revistas de investigación, los cuales han sido modificadas por el laboratorio.</div></div>

[STEPS]
TITLE: Miltenyi Biotec: Adipose Tissue Dissociation -- University of Minnesota TMCs
AUTHORS: Miltenyi Biotec, Laura J Niedernhofer, David A Bernlohr

[DESCRIPTION]
Dissociation of human adipose tissue using Miltenyi dissociation kit Catalog #130-105-808 and Protocol #140-004-708.02


[STEPS]
1.   
https://www.miltenyibiotec.com/US-en/products/adipose-tissue-dissociation-kit-mouse-and-rat.html#130-105-808
TITLE: Protocol for Lambda Exonuclease (NEB #M0262)
AUTHORS: New England Biolabs

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Lambda Exonuclease efficiently degrades 5' phosphorylated linear dsDNA from 5' to 3' direction, leaving supercoiled dsDNA intact.*</div><div class = "text-block"><span style = "font-weight:bold;">*</span><span style = "font-weight:bold;">Note: </span><span>For more precise results we recommend titration of the enzyme to the intended substrate.</span></div></div>

[STEPS]
?. Set-up the reaction as follows:  AB1Components50&nbsp;μl REACTION2&nbsp;DNA&nbsp;up to 5 μg3&nbsp;Lambda Exonuclease Reaction Buffer (10X)&nbsp;5 μl (1X)4&nbsp;Lambda Exonuclease&nbsp;1 μl (5 units)5&nbsp;Nuclease-free H2O&nbsp;up to 50 μl
AB1Components50&nbsp;μl REACTION2&nbsp;DNA&nbsp;up to 5 μg3&nbsp;Lambda Exonuclease Reaction Buffer (10X)&nbsp;5 μl (1X)4&nbsp;Lambda Exonuclease&nbsp;1 μl (5 units)5&nbsp;Nuclease-free H2O&nbsp;up to 50 μl
?. Incubate at  for .
37 °C
?. Stop reaction by adding EDTA to  .
?. Heat Inactive at  for .
75 °C
?. To clean up treated samples, we recommend using one of the following steps:a. Column clean up (we recommend the Monarch® PCR & DNA Cleanup Kit, NEB #T1030) orb. Running the reaction on an agarose gel, and then extracting the DNA (we recommend the Monarch Gel Extraction Kit, NEB #T1020), orc. Performing a phenol/chloroform extraction followed by ethanol precipitation.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
This transformation protocol is for the C2527I cells. (For the C2527H protocol, see <a href="https://www.protocols.io/view/Transformation-Protocol-C2527H-imsv4d" target="_blank">here</a>.)

[GUIDELINES]
<strong>Transformation Protocol Variables<br /></strong><br /><strong>Thawing</strong>:&nbsp;Cells are best thawed on ice and DNA added as soon as the last bit of ice in the tube disappears. Cells can also be thawed by hand, but warming above 0&deg;C will decrease the transformation efficiency.&nbsp;<br /><br /><strong>Incubation of DNA with Cells on Ice:</strong>&nbsp;For maximum transformation efficiency, cells and DNA should be incubated together on ice for 30 minutes. Expect a 2-fold loss in transformation efficiency for every 10 minutes you shorten this step.&nbsp;<br /><br /><strong>Heat Shock</strong>:&nbsp;Both the temperature and the timing of the heat shock step are important and specific to the transformation volume and vessel. Using the transformation tube provided, 10 seconds at 42&deg;C is optimal.&nbsp;<br /><br /><strong>Outgrowth</strong>:&nbsp;Outgrowth at 37&deg;C for 1 hour is best for cell recovery and for expression of antibiotic resistance. Expect a 2-fold loss in transformation efficiency for every 15 minutes you shorten this step. SOC gives 2-fold higher transformation efficiency than LB medium; and incubation with shaking or rotating the tube gives 2-fold higher transformation efficiency than incubation without shaking.<br /><br /><strong>Plating</strong>:&nbsp;Selection plates can be used warm or cold, wet or dry without significantly affecting the transformation efficiency. However, warm, dry plates are easier to spread and allow for the most rapid colony formation.

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.