eamag/protocols-with-steps huggingface dataset:
TITLE: CUMC-TMC-10X-Gene-Expression
AUTHORS: Shaunice Grier

[DESCRIPTION]
Post Mortem Human Fresh Frozen Dorsolateral Prefrontal Cortex tissues are cryo - sectioned onto 10X Visium slides.

Tissue Optimization is performed and determines optimal Gene Expression testing conditions.

cDNA samples are processed and generated into libraries. Libraries are qc'd for size and concentration with the Agilent Bioanalyzer 2100, Qubit Fluorometer and qPCR with KAPA Library Quantification Kit. QC'd libraries are submitted for sequencing.

[STEPS]
SECTION: Tissue QC
1. 
Section
SECTION: Tissue QC
2. H&E Stain
SECTION: Tissue QC
3. Image
SECTION: Tissue QC
4. Assess
SECTION: Tissue Optimization
5. Permeabilization & cDNA Synthesis

SECTION: Tissue Optimization
6. Tissue Removal
SECTION: Gene Expression
7. cDNA Synthesis
SECTION: Gene Expression
8. Second Strand Synthesis & Denaturation
SECTION: Gene Expression
9. cDNA Amplification & QC
SECTION: Gene Expression
10. Visium Spatial Gene Expression Library Construction
SECTION: Sequencing
11. Submitted libraries are evaluated for size distribution on the Fragment Analyzer and quantified using Picogreen and qPCR with the Universal KAPA Library Quantification Kit. Library pools are loaded between 1.2 – 1.5 nM using the standard NovaSeq workflow as outlined in Illumina’s NovaSeq 6000 system guide.
TITLE: Citric acid nuclei isolation from frozen oesophageal tissue 
AUTHORS: Lucy Kimbley, Rachel Parker, Jack Harrington, Robert C. Walker, Ben Grace, Jonathan J. West, Tim J. Underwood, Matthew Rose Zerilli

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Citric acid-based method of isolating nuclei from frozen oesophageal tissue. Protocol adapted from Tosti et al. [1,2] with additional optimisation for oesophageal tissue. </div><div class = "text-block">1. Tosti, L., Hang, Y., Debnath, O., Tiesmeyer, S., Trefzer, T., &  Steiger, K. et al. (2021). Single-Nucleus and In Situ RNA–Sequencing  Reveal Cell Topographies in the Human Pancreas. Gastroenterology, 160(4), 1330-1344.e11. doi: 10.1053/j.gastro.2020.11.010</div><div class = "text-block"><span>2. Luca Tosti, Christian Conrad 2020. Nuclei isolation from snap frozen human pancreatic tissue using a citric acid buffer.</span><span style = "font-weight:bold;"> protocols.io</span><a href="https://dx.doi.org/10.17504/protocols.io.tpxempn" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://dx.doi.org/10.17504/protocols.io.tpxempn</span></a></div></div>

[STEPS]
?. Weigh tissue. 30-40mg of tissue should be used per experiment. If not using the entire sample, quickly remove the tissue required by chopping sample in a petri dish chilled on dry ice (For RNA Later Ice samples see 1.1) with a cold scalpel. Return unused tissue to LN2.
?. If using tissue stored in RNA Later Ice, carry out steps 1 and 2 on wet ice. Pre-chill a petri dish on dry ice and chop tissue into very small pieces (
?. Add tissue to pre-chilled dounce homogenizer containing 2ml nuclei lysis buffer.
?. While keeping the dounce on ice, homogenize for 5 strokes using pestle A.
?. Homogenize with 5 strokes of pestle A followed by 20 strokes with pestle B. The tissue should be well homogenised and the solution in the dounce should be cloudy with no large tissue peices remaining.
?. Filter the contents of the dounce through a 30μm cell strainer. Use an additional 1ml of nuclei lysis buffer to rinse the dounce. Filter the 1ml lysis buffer through the strainer.
?. Centrifuge the nuclei suspension at 500 x g at 4°C for 5 minutes.
?. Carefully remove and discard the supernatant and resuspend the pellet in 1ml of nuclei lysis buffer.
?. Centrifuge the nuclei suspension at 500 x g, 4°C for 5 minutes. Carefully remove the supernatant and resuspend the pellet in 200μL nuclei resuspension buffer.
?. Remove 10μL of nuclei suspension and stain 1:1 with Dapi. If a high nuclei yield is anticipated, dilute the 10μL of nuclei suspension 1:5 with PBS before staining.
?. Incubate tissue on ice in the dounce for 5 minutes.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Please contact Dr. Steven Wilhelm (wilhelm@utk.edu) for additional information regarding this protocol.</p>

[STEPS]
?. 
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?. 
?. 
?.
TITLE: Single Cell Dissociation of Small Tumor Biopsies
AUTHORS: Ashley Maynard

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Dissociate small core biopies into single cells.</div></div>

[STEPS]
?. Make and/or warm Base Buffer1 to .
37 °C
?. Pour the sample(s) into a 10cm dish. Take a picture of the samples in a 10cm dish (in the media it comes in).Example:
?. Cut sample into small pieces and place into a 1.5mL tube (or multiple tubes if necessary, don’t overload the tube with tissue). Add 1.5 mL of collagenase buffer2 and digest for , , shaking in a thermomixer @ 800-1000 rpm (the tissue should not be sitting at the bottom of the tube when it’s mixing). If chunks of sample remain, pipet the sample up and down 15 times. Return the sample to the thermomixer for  at  shaking.
37 °C
37 °C
?. Remove the sample from the thermomixer. Pipet the sample up and down 15 times.
?. Filter the sample over a 100-micron filter into a new 1.5 mL tube.
?. Wash the sample filter with 1-2mL of collagenase buffer.
?. Put the cells that passed through the filter on ice.
?. Check cells with Trypan Blue and Hemocytometer (10ul Trypan blue mixed with 10ul of sample), take picture on microscope and label with date and sample ID and record # of live/dead cells.Example:
?. OPTIONAL: If there are persistent chucks of tissue that did not pass though the filter, gather them into a new 1.5 mL tube and add 0.5 mL 0.05% warmed trypsin. Digest the tissue for  at , shaking in a thermomixer @ 800-1000 rpm. Quench the reaction with 1 mL Base Buffer. Filter the sample over a 100-micron filter into a new 1.5 mL tube. Wash filter with 1-2 mL of collagenase buffer. Check cells with Trypan Blue and Hemocytometer, take picture on microscope and label with date and sample ID and record # of live/dead cells. Combine the filtered cells from step 7 and 9 into one tube.
37 °C
?. Spin at 1500 rpm for .
?. OPTIONAL (Only if pellet is visibly red): Add 0.5 mLs RBC lysis buffer to sample tubes. Leave at RT for 2-5min. Add 1.0 mL DMEM F12 + 6% FBS to each tube and spin again.Example:
?. Aspirate off supernatant. Resuspend in 120 uL of FACS Buffer3.
?. Count cells with Trypan Blue and hemocytometer (10ul Trypan blue mixed with 10ul of sample). Record the number of live and dead cells (stained blue). Take picture on microscope and label with date + sample ID + “final”
?. Remove 10ul of sample into a new 1.5mL tube, add 350uL of FACS Buffer and set aside for unstained control.
?. Stain remaining cells with 10ul CD45-FITC (if cell count is under 1x10^7) and 1ul of Hoechst stain (diluted 1:10). Incubate on ice in dark for .
?. Add 1mL of FACS Buffer to stained cells. Spin at 1500 rpm for  and aspirate off supernatant. Resuspend with 0.5 mLs of FACS Buffer.
?. before sorting add, 0.5 uL of PI and mix well. Transfer unstained and stained samples to FACS tubes and leave on ice in the dark.
TITLE: Coverage of open citations in DOAJ journals
AUTHORS: Constance  Dami, Alessandro  Bertozzi, Chiara   Manca, Umut  Kucuk

[DESCRIPTION]
This is the protocol for the research of the coverage of open citations in DOAJ journals.
Our goal is to find out:
about the coverage of articles from open access journals in DOAJ journals as citing and cited articles,
how many citations do DOAJ journals receive and do, and how many of these citations involve open access articles as both citing and cited entities,
as well as the presence of trends over time of the availability of citations involving articles published in open access journals in DOAJ journals.

[BEFORE_START]
This protocol refers to a research done for the Open Science course of 21/22.

[STEPS]
SECTION: Data Gathering
1. First, we create a script for making queries to the OpenCitations API (or SPARQL endpoints).
We make the queries on both of the indexes COCI and CROCI.

SECTION: Data Analysis
3. We do additional analysis of the data that we gathered. Maybe studying the future of the research. 
SECTION: Data Visualization
4. We visualize our results with python libraries.
SECTION: Data Gathering
1.1. First question: how many citations do DOAJ journals receive?
We count the results of a query that selects all the citations that were made to DOAJ articles.
SECTION: Data Gathering
1.2. Second question: how many citations DOAJ journals do?
We count the results of a query that selects all the citations that DOAJ articles made.
SECTION: Data Gathering
1.3. Third question: how many of these citations involves articles published in open access journals as both citing and cited entities?
We take the results of the previous queries and we check, with the use of the metadata of the DOIs through the OpenCitations API, if they are citations from open access journals.

SECTION: Publishing data
2. We publish the data that we gathered in JSON and CSV.
SECTION: Data Gathering
1.4. Fourth question: what is the trend (increasing? decreasing?) in time of the availability of such citations involving journals in DOAJ?
We take the previous results and group them in years.
TITLE: Protocols for &#34;Synaptotagmin-1-dependent phasic axonal dopamine release is dispensable for basic motor behaviors in mice&#34;
AUTHORS: Benoît Delignat-Lavaud, Jana Kano, Charles Ducrot, Ian Massé, Sriparna Mukherjee, Nicolas Giguère, Luc Moquin, Catherine Lévesque, Samuel Burke, Raphaëlle Denis, Marie-Josée Bourque, Alex Tchung, Pedro Rosa-Neto, Daniel Lévesque, Louis De Beaumont, louis-eric.trudeau

[DESCRIPTION]
In Parkinson’s disease (PD), motor dysfunctions only become apparent after extensive loss of DA innervation. This resilience has been hypothesized to be due to the ability of many motor behaviors to be sustained through a diffuse basal tone of DA; but experimental evidence for this is limited. Here we show that conditional deletion of the calcium sensor synaptotagmin-1 (Syt1) in DA neurons (Syt1 cKODA mice) abrogates most activity-dependent axonal DA release in the striatum and mesencephalon, leaving somatodendritic (STD) DA release intact. Strikingly, Syt1 cKODAmice showed intact performance in multiple unconditioned DA-dependent motor tasks and even in a task evaluating conditioned motivation for food. Considering that basal extracellular DA levels in the striatum were unchanged, our findings suggest that activity-dependent DA release is dispensable for such tasks and that they can be sustained by a basal tone of extracellular DA. Taken together, our findings reveal the striking resilience of DA-dependent motor functions in the context of a near-abolition of phasic DA release, shedding new light on why extensive loss of DA innervation is required to reveal motor dysfunctions in PD.

[STEPS]
TITLE: Immunohistochemistry of free floating slices
AUTHORS: louis-eric.trudeau

[DESCRIPTION]
This protocol details the immunohistochemistry of free floating slices.

[STEPS]
SECTION: Steps
1. 
 

Fix with paraformaldehyde (PFA) 4%   at Room temperature.
- 4 g of PFA
- Dissolve in 100 mL de PBS (pH pH 7.3) previously heated to around 60 °C.
- Mix the solution until it completely clears up.
- Filter and store at 4 °C for a duration of 20160 min.
SECTION: Steps
3. Permeabilize and block the non-specific primary antibodies bindings in a 24-well multiwell plate
by incubating under agitation for 60 min with 0.5 mL to 1 mL of a BSA enriched ab solution (10 mL of ab solution for 1 g of BSA).
SECTION: Steps
4. All the subsequent steps are done using the following ab solution:

- 95 mL of PBS pH 7.3
- 20 mg of NaN3 (final 0.02 %)
- 300 µL of Triton X-100 (final 0.3 %)
- 500 mg of BSA (final 0.5 %)
- 5 mL of goat serum (final 5 %)
SECTION: Steps
5. Dilute the primary antibodies in the ab solution and apply 0.5 mL per well (up to 10 slices) 
60 min at Room temperature under agitation.
SECTION: Steps
6. Rinse the slices with PBS (3X 10 min) under agitation.
SECTION: Steps
7. Dilute the secondary antibodies in the ab solution and apply 0.5 mL per well (up to 10 slices) for 120 min at Room temperature under agitation.
SECTION: Steps
8. Rinse the slices with PBS (3X 10 min) under agitation.
SECTION: Steps
9. Mount the slices on charged (+) glass slides submerged in PBS.
SECTION: Steps
10. Let the mounted slices dry (about 10 min). 

 

SECTION: Steps
11. Seal the slides with 100 µL-120 µL/slide of Fluoromount G and a rectangular glass coverslip and seal the coverslip to the slide with nail polish.
SECTION: Steps
2. Rinse with PBS 1X (3X 10 min) under agitation.
SECTION: Steps
2.1. Rinse with PBS 1X for 10 min under agitation (1/3).
SECTION: Steps
2.2. Rinse with PBS 1X for 10 min under agitation (2/3).
SECTION: Steps
2.3. Rinse with PBS 1X for 10 min under agitation (3/3).
SECTION: Steps
6.1. Rinse the slices with PBS for 10 min under agitation (1/3).
SECTION: Steps
6.2. Rinse the slices with PBS for 10 min under agitation (2/3).
SECTION: Steps
6.3. Rinse the slices with PBS for 10 min under agitation (3/3).
SECTION: Steps
8.1. Rinse the slices with PBS for 10 min under agitation (1/3).
SECTION: Steps
8.2. Rinse the slices with PBS for 10 min under agitation (2/3).
SECTION: Steps
8.3. Rinse the slices with PBS for 10 min under agitation (3/3).
TITLE: Nielsen and Ford (2023) M4-mediated cholinergic transmission is reduced in parkinsonian mice and its restoration alleviates motor deficits and levodopa-induced dyskinesia
AUTHORS: Beatriz E Nielsen, Christopher P Ford

[DESCRIPTION]
This collection contains protocols detailing methods used in Nielsen and Ford (2023) M4-mediated cholinergic transmission is reduced in parkinsonian mice and its restoration alleviates motor deficits and levodopa-induced dyskinesia




[STEPS]
TITLE: Molecular cloning of SHIP164 plasmids for expression in mammalian cells
AUTHORS: michael.hanna , Michael G Hanna, Pietri De Camilli

[DESCRIPTION]
This protocol is to help with the molecular cloning of SHIP164 and the sequences of other proteins.

[BEFORE_START]
We experienced difficulty in amplifying SHIP164 from a cDNA library, therefore we opted to generate a codon
optimized SHIP164 open reading frame synthesized. The codon optimized cDNA was cloned into pUC57 and was
subsequently subcloned into multiple other mammalian expression vectors.

The general cloning strategy is described below.

[STEPS]
SECTION: Cloning strategy
1. Linearize destination vector using restriction enzyme digest following manufacturers protocol (New England Biolabs).
SECTION: Cloning strategy
2. Amplify desired insert by PCR making sure to include 15-20 bp overhang homology with destination vector.
SECTION: Cloning strategy
3. Run both cut vector and PCR product on an agarose gel (e.g., 1% supplemented with Midori Green or Ethidium Bromide for visualization) and purify the DNA from gel using a NucleoSpin Gel and PCR Clean-up kit (Takara).
SECTION: Cloning strategy
4. Ligate PCR insert into vector using NEB HiFi assembly mixture following manufacturers protocol
(https://www.neb.com/protocols/2014/11/26/nebuilder-hifi-dna-assembly-reaction-protocol).
TITLE: Sidoli Phosphoenrichment From Clean Dry Peptides
AUTHORS: Edwin Yoo

[DESCRIPTION]
Sidoli Phosphoenrichment From Clean Dry Peptides

[STEPS]
1. Obtain trypsinized peptides that are clean and dried (ie. completed desalting or S-trap).
 

2. - Dilute your peptide solution at least 10 times with loading buffer (alternatively if you have 100 μL sample you can add 50 μl water, 50 μL 100% TFA, 800 μL Acetonitrile and 76 mg Glycolic acid to make the sample up to the proper loading buffer). If completely dry, can just resuspend with 100 ul loading buffer.
3. - Add 0.6 mg TiO2 beads per 100 μg peptide solution. Make up enough master mix of bead suspension.
 
 
4. - Place the tubes on the shaker (highest shaking) at room temperature for 5-10 min - Centrifuge to pellet the beads (table centrifuge <15 sec)
5. - Remove the supernatant
6. - Wash the beads with 70-100 μL loading buffer - mix for 15 sec, transfer to another eppendorff tube and then centrifuge to pellet the beads (the transfer is performed due to the fact that peptides and phosphopeptides stick to surfaces and can be eluted in the last elution step)
7. - Wash with 70-100 μL washing buffer 1 - mix for 15 sec and then centrifuge to pellet the beads. Remove supernatant.
8. - Wash with 50-100 μL washing buffer 2 - mix for 15 sec and then centrifuge to pellet the beads. This step is important to remove peptides that bind in a HILIC mode to TiO2. Remove supernatant.
9. - Dry the beads for 5-10 min in the vacuum centrifuge or on the table.
10. - Elute the phosphopeptides with 50 μL Elution buffer – mix well and leave the solution and the beads for 5-10 min to allow an efficient elution
11. - Centrifuge the solution for 1 min, recover the supernatant (as it contains the phosphopeptides) and dry it in a SpeedVac centrifuge. Ready for injection.
TITLE: Low Cell Input Nuclei Isolation for Single Cell ATAC-Seq
AUTHORS: Carmen Sancho

[DESCRIPTION]
This protocol describes the the methodology of nuclei isolation for Single Cell ATAC Sequencing adopted from 10X Genomics guide CG000169 Revision D. 

[STEPS]
SECTION: Prepare diluted nuclei buffer
1. 
Keep nuclei buffer on ice! 
 

SECTION: Prepare buffer A
2. Keep buffer A on ice and prepare fresh!  
 

SECTION: Prepare wash buffer
3. Keep wash buffer on ice and prepare fresh!  
 

SECTION: Additional buffers
5. PBS + 0.04% BSA (maintain at 4 °C )

 

SECTION: Nuclei isolation from fresh cells
6. Nuclei may be isolated from 2000-100,000 cells using this protocol. Cells are already resuspended in PBS + 0.04% BSA. 

Centrifuge cell suspension at 300 rcf for 5min at 4 °C  and re-suspend the cell pellet in 50 ul of PBS 0.04% BSA. Transfer 50 ul cell suspension to a 0.2ml tube. Proceed to next step. 

SECTION: Nuclei isolation from fresh cells
7. Centrifuge at 300 rcf for 5 min at 4 °C. 


SECTION: Nuclei isolation from fresh cells
8. Remove 45ul supernatant without touching the bottom of the tube to avoid dislodging the cell pellet. 
SECTION: Nuclei isolation from fresh cells
9. Add 45 µLchilled Lysis Buffer. Gently pipette mix 3x. 

SECTION: Nuclei isolation from fresh cells
10. Incubate for 4 min* on ice. 

*Time between 3-5min, depending on cell type.
SECTION: Nuclei isolation from fresh cells
11. Add 50 µL chilled Wash Buffer to the tube. DO NOT MIX!

SECTION: Nuclei isolation from fresh cells
12. Centrifuge at 500 rcf for 5 minat 4 °C. 


SECTION: Nuclei isolation from fresh cells
13. Remove 95ul supernatant without disrupting the nuclei pellet. 
SECTION: Nuclei isolation from fresh cells
18. **If original cell count <20,000 cells, do not need to count nuclei. Load everything into 10X. 


Use 2.5 µL nuclei suspension mix with 2.5 µL Diluted Nuclei Buffer and 5 µL  Trypan blue to determine cell count. 


nuclei count= _____ x 2 x 2 x 10 nuclei/ul
SECTION: GEMS generation
24. Proceed immediately to Chromium Single Cell ATAC Solution User Guide PAGE 24

CG000168_ChromiumSingleCell_ATAC_ReagentKits_UserGuide_RevC.pdf


SECTION: Nuclei isolation from fresh cells
14. Add 45 µL chilled Diluted Nuclei Buffer to the pellet. DO NOT MIX. 

SECTION: Nuclei isolation from fresh cells
15. Centrifuge at 500 rcf for 5 minat 4 °C. 
SECTION: Nuclei isolation from fresh cells
16. Remove the supernatant in 2 step without touching the bottom of the tube to avoid dislodging the nuclei pellet. 


SECTION: Nuclei isolation from fresh cells
17. Resuspend the nuclei pellet in 5.5 µL chilled diluted nuclei buffer (pellet may not be visible).

SECTION: Nuclei isolation from fresh cells
19. 
 
SECTION: Prepare transposition mix
22.  
SECTION: Nuclei isolation from fresh cells
20.  

SECTION: Prepare transposition mix
23.  

SECTION: Nuclei isolation from fresh cells
21. Proceed to load about 5 µL  of nuclei suspension for transposition step.

SECTION: Prepare lysis buffer
4. Keep lysis buffer on ice and prepare fresh!
TITLE: Testing platelet aggregation activity
AUTHORS: Jamilya Mansurova  , Ludmila Karazhanova, Aisulu Zhunuspekova

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Aggregatometry is based on the ability of platelets to activate and form cell aggregates under the influence of inductors. During the analysis, changes in the light transmission of platelet-rich plasma are recorded as a result of the interaction of platelets through the IIb / IIIa glycoprotein receptors in response to stimulation by the inducer. The work of optical aggregometer is based on this principle. When assessing platelet aggregation according to G. Born and J. O'Brien, platelets are activated at 37°C by adding one of the agonists (1,2). Then, platelet aggregation occurs and transparency in the blood sample increases, which leads to an increase in the aggregation curve.</div></div>

[STEPS]
?. Whole blood samplingBlood sampling was performed in 12-48 hours after PCI in the morning on an empty stomach from the cubital vein into vacuum tubes with 3.8% sodium citrate (in a ratio of 9: 1) with short-term application of a tourniquet and with 0.8 mm. diameter needle. The blood samples were delivered to the laboratory within 20-30 minutes.
?. Platelet-rich plasma productionTo obtain platelet-rich plasma, the test tube with blood was rotated on the OPn-3.02 "Dastan" centrifuge (Kyrgyzstan) at 1000 rpm within 10 minutes.
?. Platelet-poor plasma productionThe tube with platelet-rich plasma was re-centrifuged at 3000 rpm for 15 minutes.
?. Determining residual platelet reactivityThe antithrombotic efficacy of antiplatelet agents was tested by an AggRAM Helena Biosciences Europe aggregometer using adenosine-5`-diphosphate (ADP) and epinephrine by Tekhnologiya-Standard inducer (Russia) at a dilution of 10 μg / ml and 2.5 μg / ml. respectively. The aggregation registration time was 10 minutes. The results of the study of platelet function were assessed by the maximum percentage of aggregation (max%) in response to stimulation with the inductor and the area under the aggregation curve (AUC – area under curve). Platelet-poor plasma was regarded as 100%, while platelet-rich plasma was taken as 0%.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
For use in the protocol "<a href="https://www.protocols.io/view/Large-Volume-Marine-Cyanophage-Phage-Protocols-c3iykd" target="_blank">Large Volume Marine Cyanophage protocols</a><span data-sheets-value="[null,2,&quot;Large Volume Marine Cyanophage protocols&quot;]" data-sheets-userformat="[null,null,513,[null,0],null,null,null,null,null,null,null,null,0]">"</span>

[STEPS]
?. 
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Please contact Dr. Steven Wilhelm (wilhelm@utk.edu) for additional information regarding this protocol.</p>

[STEPS]
?. 
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TITLE: SDS-PAGE
AUTHORS: Alexander Röntgen

[DESCRIPTION]
Protocol for running an SDS-PAGE.

[STEPS]
SECTION: Sample preparation
1. Dilute NuPAGE Sample Buffer 4X Concentrate into protein samples 


SECTION: Sample preparation
2. Heat for 10 min at 95 °C 
SECTION: Gel
3. Run gel at ~150 V for 35 min
SECTION: Gel
4. Incubate in staining solution until bands appear
SECTION: Gel
5. Image using ChemiDoc MP Imaging system