eamag/protocols-with-steps huggingface dataset:
TITLE: Staining of GFAP and IBA1 in PDGFR-B/Td-tomato brain sections
AUTHORS: Daniel Manrique-Castano

[DESCRIPTION]
This protocol is suitable for the staining of GFAP and IBA1 in PDGFR-B/Td-Tomato fixed mouse brain sections.

[GUIDELINES]
Read the entire protocol before starting the procedure.

Note that this protocol uses  3 hours (room temperature) incubation for primary antibodies. 

Do not submit the tissue to antigen retrieval or other acidic solutions to preserve theTd-tomato signal.

Works with the listed antibodies and dilutions. For other references, preliminary tests are highly recommended.   


[STEPS]
SECTION: Tissue preparation and blocking
1. Take out sections from -80 and place them in an incubator/plate for 20 min  at 37 °C. This step is performed to ensure tissue attachment to the crystal slides. 

SECTION: Tissue preparation and blocking
2. 2. Draw a hydrophobic barrier on each slide using ImmEdge®Hydrophobic Barrier PAP Pen and let dry for about 10 min minutes. This will prevent buffers from escaping the tissue area.
 


SECTION: Tissue preparation and blocking
3. To initially rehydrate and permeabilize the tissue, place the slides in a slide jar containing the permeabilization solution with shaking for 30 min
 
.

SECTION: Tissue preparation and blocking
4. To prevent unspecific binding, incubate brain sections in permeabilization solution containing 5 % (v/v)    and 1 Mass / % volume  for 60 min at Room temperature.
 



SECTION: Antibody incubation
5. When blocking is finished, decant the buffer (no washing is required) and incubate primary antibodies in antibody buffer for 180 min at Room temperature according to table 1.


 

 

SECTION: Antibody incubation
6. When primary antibody incubation is finished, wash the sections 5 min with 0.05 % (v/v)    in PBS.
SECTION: Antibody incubation
7. Incubate secondary antibodies in 0.1 % (v/v)      for 60 min at Room temperature according to table 2.
 
 

SECTION: Antibody incubation
8. When secondary antibody incubation is finished, wash the sections 5 min with 0.05 % (v/v)    in PBS. Follow this with 5 min washes with PBS to remove all detergent traces.  

SECTION: Antibody incubation
9. Clean the remaining buffer on the slides using absorbent tissue and mount the sections with a drop flo .
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Given a set of temporal networks, from different domains and with different sizes, how can we compare them? Can we identify evolutionary patterns that are both (i) characteristic and (ii) meaningful? We address these challenges by introducing a novel temporal and topological network fingerprint named Graphlet-orbit Transitions (GoT). We demonstrate that GoT provides very rich and interpretable network characterizations. Our work puts forward an extension of graphlets and uses the notion of orbits to encapsulate the roles of nodes in each subgraph. We build a transition matrix that keeps track of the temporal trajectory of nodes in terms of their orbits, therefore describing their evolution. We also introduce a metric (OTA) to compare two networks when considering these matrices.</p>
<p> </p>
<p>Our experiments show that networks representing similar systems have characteristic orbit transitions. GoT correctly groups synthetic networks pertaining to well-known graph models more accurately than competing static and dynamic state-of-the-art approaches by over 30%. Furthermore, our tests on real-world networks show that GoT produces highly interpretable results, which we use to provide insight into characteristic orbit transitions.</p>

[BEFORE_START]
<p>A version of GoT with set of temporal networks is available at http://www.dcc.fc.up.pt/got-wave/download.</p>
<p> </p>
<p>GoT was written and C++ and compiled in Fedora 26 and Ubuntu 16 LTS.</p>

[STEPS]
?. 
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TITLE: Pre-sessions description
AUTHORS: Ana  Rachel Pinto, Rita  Matos Sousa, Maria  Margarida Teles Vasconcelos Correia Neves, Joana  Almeida Santos Pacheco Palha, José  Miguel Moreira Pêgo, Nuno  Carvalho Sousa, Paul  Scoles, Peter  Vincent Scoles

[DESCRIPTION]
This is a step-by-step description of the content of the pre-sessions which was focused on clarifying concepts, explaining the case-based learning sessions sequences.

[STEPS]
TITLE: Endometrium - Collagenase 
AUTHORS: Roser Vento-Tormo, Regina Hoo

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol describe the tissue dissociation procedures from human endometrium and pregnancy endometrium samples. This protocol is adapted from</span><span style = "font-style:italic;"> Vento-Tormo et al. 2018 Nature</span><span> with some modification from Prof. Ashley Moffett (Department of Pathology, University of Cambridge). </span></div></div>

[STEPS]
?. [Tissue dissociation and digestion]
Note: Flash-frozen tissue with isopentane for Spatial Transcriptomics work. {optional} Note: If tissue is going to be transported, do it with preservation solution (HypoThermosol® FRS) at  . Store sample for fixing in formalin (RNA Scope) & nuclei sequencing (flash-frozen) {optional}.Scrape off the blood vessels on remaining tissues. Note: We can skip this step if the donor is perfused.
4 °C
?. [Tissue dissociation and digestion]
Place wet tissue under a petri dish. Take 2 scalpels and roughly mince up the tissue. This step is crucial to increase the efficiency of the digestion.
?. [Tissue dissociation and digestion]
Transfer contents to 50ml falcon containing the collagenase mix (~  /tissue but it will depend on the size of the tissue)
3 ml
?. [Prepare collagenase mix]
Collagenase mix recipe:   ABCD1ProductStockFinal volume (20ml : 3ml/sample)Concentration2RPMI or Hams F12 + 10% FBS9 ml RPMI or Ham's F12 + 1 ml FBS8.9 ml3Collagenase V10 mg/ml1 ml1 mg/ml4DNase I10 mg/ml100 ul0.1 mg/ml
ABCD1ProductStockFinal volume (20ml : 3ml/sample)Concentration2RPMI or Hams F12 + 10% FBS9 ml RPMI or Ham's F12 + 1 ml FBS8.9 ml3Collagenase V10 mg/ml1 ml1 mg/ml4DNase I10 mg/ml100 ul0.1 mg/ml
?. [Tissue dissociation and digestion]
Tighten lid and then seal with parafilm.
?. [Tissue dissociation and digestion]
Incubate at  for  . Shacking during the incubation is recommended.
37 °C
?. [Tissue dissociation and digestion]
Resuspend with   RPMI 10%.
20 ml
?. [Tissue dissociation and digestion]
Filter sample through small strainer (100um) – do not discard retained tissue. The retained tissue will be used for "Endometrium-Trypsin" protocol.
?. [Tissue dissociation and digestion]
Filtered material: Centrifuge at 450 g,   (0.5 rcf,  ).
?. [Tissue dissociation and digestion]
Wash with  of PBS twice.
10 ml
?. [Tissue dissociation and digestion]
Resuspend with RPMI 10%, count cells and sort for cell population (refer to "Cell staining for flow cytometry and sorting" protocol)
1 ml
?. [Tissue dissociation and digestion]
{optional- for biopsies we do this} Resuspend sample with  to  of 1X RBC lysis buffer mix and incubate for . *RLB preparation: Dilute 10X RLB stock with water. After RBC lysis, add of RPMI 10% and centrifuge 450g for Wash twice with 10ml of PBS.
2 ml
4 ml
10 ml
?. [Tissue dissociation and digestion]
{Optional} Resuspend cells in freezing medium to a concentration of 1x 107  cells and aliquot into cryogenic storage vials.
?. [Tissue dissociation and digestion]
Wash tissue with PBS {optional}.
TITLE: Collection: State-of-the-Art Analytical Methods of Viral Infections in Human Lung Organoids
AUTHORS: Morris Baumgardt, Maren Hülsemann, Anna Löwa, Diana Fatykhova, Karen Hoffmann, Mirjana Kessler, Maren Mieth, Katharina Hellwig, Doris Frey, Alina Langenhagen, Anne Voss, Benedikt Obermayer, Emanuel Wyler, Simon Dökel, Achim D. Gruber, Ulf Tölch, Stefan Hippenstiel, Andreas C. Hocke, Katja Hönzke

[DESCRIPTION]
Organ models have received widespread attention in the study of SARS-CoV-2, the pathogen causing the current COVID-19 pandemic. Human-based organ models can provide strong predictive value to investigate the tropism, virulence, and replication kinetics of viral pathogens.
Applicable to a large set of organoid models and viruses, we provide a step-by-step work instruction for the infection of human alveolar-like organoids with SARS-CoV-2 in this protocol collection. We also prepared a detailed description on state-of-the-art methodologies to assess the infection impact and the analysis of relevant host factors in organoids.
This protocol collection consists of five different sets of protocols. Set 1 describes the protein extraction from human alveolar-like organoids and the determination of protein expression of angiotensin-converting enzyme 2 (ACE2), transmembrane serine protease 2 (TMPRSS2) and FURIN as exemplary host factors of SARS-CoV-2. Set 2 provides detailed guidance on the extraction of RNA from human alveolar-like organoids and the subsequent qPCR to quantify the expression level of e.g., ACE2 or other host factors of SARS-CoV-2 on RNA level. Protocol set 3 contains an in-depth explanation on how to infect human alveolar-like organoids with SARS-CoV-2 and how to quantify the viral replication by plaque assay and viral E gene-based RT-qPCR. Set 4 provides a step-by-step protocol for the isolation of single cells from infected human alveolar-like organoids for further processing in single-cell RNA sequencing or flow cytometry. Set 5 presents a detailed protocol on how to perform the fixation of human alveolar-like organoids and guides through all steps of immunohistochemistry and in situ hybridization to visualize SARS-CoV-2 and its host factors. The infection and all subsequent analytical methods have been successfully validated by biological replications with human alveolar-like organoids based on material from different donors.

[BEFORE_START]
Grow the virus stock (SARS-CoV-2 B.1) on Vero E6 cells (RRID:CVCL_0574), please work with maximum passage 3 and sequence the virus stock initially. 

[GUIDELINES]
This protocol collection describes the processing of human alveolar-like organoids which have been grown according to Youk et al., 2020. https://doi.org/10.1016/j.stem.2020.10.004.

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<div class="row bibliography-item-info">
<div class="bibliography-item-copy-text content col-md-12" data-clipboard-target="copy-target-228943496" data-redirect-target="/items/228943496/copy">Van Etten, J. (n.d.). Formulation of Modified Bold's Basal Medium (MBBM). Retrieved from http://ncv.unl.edu/vanettenlab/</div>
<div class="bibliography-item-copy-text content col-md-12" data-clipboard-target="copy-target-228943496" data-redirect-target="/items/228943496/copy">
<div>
<div>
<div data-clipboard-target="copy-target-228943496" data-redirect-target="/items/228943496/copy">Contact Dr. Steven Wilhelm (wilhelm@utk.edu) or Samantha Coy (srose16@vols.utk.edu) for additional information regarding this protocol.</div>
</div>
</div>
<div> </div>
</div>
</div>
<div class="row bibliography-item-actions"> </div>

[STEPS]
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?.
TITLE: Qubit4 Fluorometer Protocol: Invitrogen dsDNA HS Assay Kit (REF 32854)
AUTHORS: Jenna Brown, Mirayana Marcelino Barros, Tiago Pereira, Alejandro De Santiago Perez, Hunter Powell

[DESCRIPTION]
Qubit4 Fluorometer Protocol: Invitrogen dsDNA HS Assay Kit (REF 32854); Qubit4 protocol used to test dsDNA concentrations in samples.

[STEPS]
2. Prepare standards separately (Standard #1 and Standard #2):  190 µL of working solution and 10 µL of standard,  to a final volume of 200 µL for each standard. 
Tip: Slowly mix up and down with pipette tip.
3. Prepare samples:  198 µL of working solution and 2 µL of extracted DNA, to a final volume of 200 µL for each sample. 
Tip: When pipetting DNA, make sure to mix slowly with pipette tip to prevent shearing. Slowly mix this mixture up and down with pipette tip as well. 

4. Vortex all tubes and centrifuge briefly. Remove any bubbles before inserting tubes into the Qubit4 Fluorometer.

6. Insert standard tubes into the Qubit Fluorometer and take readings. Note: make sure that standard readings are accurate before moving on to the samples. (Standard #1: low, Standard #2: high). Specify the volume of DNA used and then take readings of your samples. 


SECTION: Qubit4 Fluorometer Protocol: Invitrogen dsDNA HS Assay Kit (REF 32854)
1.1. Preparing working solution:
Dilute the Qubit reagent 1:200 in Qubit buffer: 1 µL of Qubit dsDNA HS Reagent *200X (multiplied by number of samples + one extra sample) for every 199 µL of Qubit dsDNA HS Buffer (multiplied by number of samples + one extra sample). Make enough mix for the total number of samples including Standard #1 and Standard #2.
SECTION: Qubit4 Fluorometer Protocol: Invitrogen dsDNA HS Assay Kit (REF 32854)
1. Set up two assay tubes (Qubit assay tubes REF Q32856) for the standards (Standard #1 and Standard #2) and one assay tube for each sample.
5. Setting up the Qubit4 Fluorometer: (a) select dsDNA, (b) choose dsDNA HS.
TITLE: 12 Ion Exchange
AUTHORS: TJUSLS China

[STEPS]
?. According to the predicted pI of the protein and the pH of the ion-exchange column buffer, firstly select the appropriate ion exchange column (anion exchange column or cation exchange column). The pH of buffer should deviate from the isoelectric point of the protein.
?. The protein is concentrated with a 10KD concentration tube, and then the exchange buffer is used to exchange the protein to the ion-exchange liquid A. Finally, it is concentrated to less than 5ml by centrifuging at 4℃ and 3400rpm for 10 minutes in a high-speed centrifuge to remove insoluble substances and bubbles.
5 ml
4 °C
?. Balance the selected column with liquid A.
?. Through the AKTApure protein purification system, the samples are loaded to the column at a flow rate of 0.5ml/min, and continue washing for 5min.
?. Gradually increase the content of liquid B in the column, change the salt concentration and then change the interaction between the sample and the column, and collect the corresponding eluent according to the position of the peak.
?. Use SDS-PAGE to check the result.
TITLE: Microfluidics 1 - Mold Fabrication: Spin Coating of Photoresist
AUTHORS: Serhat S,  C. Yunus Sahan

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:left">Microfluidic Lab-on-a-chip technology has several different materials and fabrication methods. PDMS (PolyDiMethylSiloxane) is a well-known material and due to its several advantages, it is one of the most prefered material. PDMS chip fabrication technique requires an initial master mold on which replicates can be done. Su8 is a photoresist resin used in MEMS technology for relatively thick structures. PDMS microfluidic chips fabrication is suitable for SU8+Si wafer molds. This protocol describes the coating of SU8 photoresist on silicon wafers by spin coating method in our laboratories.</div></div><div class = "text-block"><div class = "justify" style = "text-align:left"></div></div></div>

[STEPS]
?. [SpinCoater Instrument Adjustment]
Dispense 1ml of photoresist (SU8) resin for each inch (25mm) of Si wafer substrate diameter.Spin at 500 rpm for 10 seconds with an acceleration of 100 rpm/second.Spin at "N" rpm for 30 seconds with an acceleration of 300 rpm/second.Expected results of photoresist thickness:   40um film thickness with N=4000 rpm spin of SU8 2050
   25um film thickness with N=4000 rpm spin of SU8 2025
   75um film thickness with N=2000 rpm spin of SU8 2050
   40um film thickness with N=2000 rpm spin of SU8 2025
The step is performed inside the Class 100.000 cleanroom and in a fume hood.
?. [Spin Coater Run]
Place the wafer in the spin coater and run the device.Microscope slides, Si/SiO2 wafers, and glass/PMMA wafers are alternatives substrates.
?. [Soft Baking of SU8 Coated Wafer]
Soft baking is done depending on the thickness of the coated photoresist film.It is a few minutes of baking at 65oC and 5-15 minutes of baking at 95oC are applied.
?. [Stocking the Coated Wafer]
In a petri plate, covered tightly with aluminum foil, photoresist-coated wafers can be stored in the cleanroom at room temperature for approximately one month.
TITLE: Suggested protocol for loading a DNA Ladder/marker
AUTHORS: New  England   Biolabs

[DESCRIPTION]
This is the suggested protocol for use with: 

Quick-Load®Purple 1 kb DNA Ladder (N0552)
Quick-Load®Purple 100 bp DNA Ladder (N0551)
Quick-Load®Purple 50 bp DNA Ladder (N0556)
Quick-Load®1 kb Extend DNA Ladder (N3239)
Quick-Load®1 kb DNA Ladder (N0468)
Supercoiled DNA Ladder (N0472S)
λ DNA-Mono Cut Mix (N3019)
фX174 DNA-HaeIII Digest (N3026)
pBR322 DNA-BstNI Digest (N3031)
pBR322 DNA-MspI Digest (N3032)
2-Log DNA Ladder (0.1-10.0 kb) (N3200)
100 bp DNA Ladder (N3231)
1 kb DNA Ladder (N3232)
Low Molecular Weight DNA Ladder (N3233)
50 bp DNA Ladder (N3236)

[BEFORE_START]
This protocol is recommended for a 5 mm wide gel lane. The components of the mixture should be scaled up or down, depending on the width of the lane.

[STEPS]
1. Prepare loading mixture (6 μl total volume):   
 


 

2. Mix gently by pipetting.
3. Load onto the agarose gel.
TITLE: Protein Expression
AUTHORS: Rengin Yamur Akbiyik, Sevval Uysalcan

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The protein expression is a process about synthesizing and modification for proteins in living</div><div class = "text-block">organisms with using several laboratory techniques and devices. This technique is generally</div><div class = "text-block">used for obtaining protein from the competent cell that is used for carrying plasmids that possess</div><div class = "text-block">the protein that we want to express.</div><div class = "text-block"><span style = "font-weight:bold;">Protein expression involves two general steps that;</span></div><div class = "text-block">• Gene expression; which is continuum that converting DNA to the mRNA that can be</div><div class = "text-block">translate into protein.</div><div class = "text-block">• Protein manufacturing; which is translation process.</div></div>

[STEPS]
?. [Transform BL21 cells and plate them on Amp (Kan).]
?. [Afer overnight incubation, take colonies and transfer into 6 ml LB with 6 µl Amp. Let them grow for 3-5 hours.]
[LB]
[Ampicillin]
?. [When incubation process is done, transfer it to 1 L ZYM-5052 Medium with 1 µl Amp.]
?. [Centrifuge at 6000 rpm for 10 minutes.]
[Centrifuge]
?. [Discard liquid and repeat centrifuge step for 3 times total.]
[2nd Centrifuge]
[3rd Centrifuge]
?. [Add Binding Buffer.]
[Binding Buffer]
?. [Transfer it to 50 ml falcon tube.]
In this step 35 µl of total sample should be obtained. Binding Buffer involves 5 ml Imidazole, 20 ml TBS, ddw up to 200 ml.
?. [For the resuspension of pallet, tubes with Binding Buffer can be put into shaker.]
?. [Sonication will be occured.]
For sonication, plastic test tubes are usually used for sonication. This sonication process is done at 3 cycles for 7 minutes. Repeat this step for 3 times total.
[Sonication for 1 cycle.]
?. [Centrifuge at 13000 rpm for 30 minutes.]
[Centrifuge]
TITLE: Genetic interaction analysis using ERGs in D. melanogaster
AUTHORS: Natalie Kaempf, Ayse Kilic, Patrik Verstreken

[DESCRIPTION]
This protocols describes the recording of electroretinograms (ERGs) and calculation of genetic interactions of fly mutants for a transheterozygous screen.

[STEPS]
SECTION: Electroretinograms (ERGs)
1. For every measurement male flies are collected (including the control) 4±1 d after eclosion from the transheterozugous crosses reared in a temperature and light-controlled incubator
at 25 °C and 12-hour light-dark cycle 
SECTION: Electroretinograms (ERGs)
2. Flies are sedated with CO2 and immobilized on glass microscope slides using double-sided tape
SECTION: Electroretinograms (ERGs)
4. Glass electrodes (borosilicate, 1.5 mm outer diameter) filled with 3 M NaCl are placed in the thorax as a reference and on the fly eye for recordings
SECTION: Electroretinograms (ERGs)
5. The flies are exposed to darkness for 3 seconds, followed by 1 second of LED light illumination inside a Faraday Cage.
SECTION: Electroretinograms (ERGs)
6. This is repeated 4 times for each fly (5 times in total).
SECTION: Electroretinograms (ERGs)
7. Light-evoked signals are amplified by a DC amplifier and the amplified signal is processed
by a data acquisition device (Clampex) and Axosope 10.7, connected to a PC running Clampfit 10.7 software (Molecular Devices). 
SECTION: Electroretinograms (ERGs)
8. First control flies are recorded to to assess whether the recording is stable and conditions are comparable to previous recordings
SECTION: Analysis for genetic interaction using non-interacting model
10. For all ERGs the depolarization amplitude is determined and normalized to the mean depolarization amplitude of the control of the individual experiment.
SECTION: Analysis for genetic interaction using non-interacting model
11. The expected depolarization amplitude for every gene pair is calculated by multiplying the normalized mean depolarization amplitude of the single heterozygous mutants.
SECTION: Analysis for genetic interaction using non-interacting model
12. The genetic interaction strength is calculated by subtracting the modelled expected depolarization (step 9) from the individual observed depolarization amplitude of double heterozygous gene pairs
SECTION: Electroretinograms (ERGs)
9. ERG traces are analyzed for depolarisation amplitude and On/Off peaks in IGOR Pro 6.37 (WaveMetrics) using a custom-made macro.
SECTION: Electroretinograms (ERGs)
3. Filamented glass micropipettes are used to generate electrodes using the Laser-pipette-puller P2000.
TITLE: U54 SCENT 10x Genomic Single-Cell Multiome
AUTHORS: Karen Abramson, Simon Gregory

[DESCRIPTION]
This document outlines the 10X Genomics Single-Cell Multiome Protocol used for normal lung and colon tissues for SCENT at Duke University.

[STEPS]
SECTION: 10X Gemonics Single-Cell Multiome Protocol
1. Nuclei were isolated using this 10x Genomics kit - Chromium
Nuclei Isolation with RNase Inhibitor Kit, 16rxns, and this protocol

SECTION: 10X Gemonics Single-Cell Multiome Protocol
2. Multiome libraries were generated using these kits -
Chromium Next GEM Single Cell Multiome ATAC + Gene Expression Reagent Bundle and this protocol

SECTION: 10X Gemonics Single-Cell Multiome Protocol
3. Libraries were sequenced on Illumina sequencers to a read depth of at least 50k reads/nucleus for Gene Expression with read lengths of 28x10x10x90 and at least 25k reads/nucleus for ATAC with read lengths of 50x8x24x49.