eamag/protocols-with-steps huggingface dataset:
TITLE: Transient transformation of  Ostreococcus species (OTTH595,  RCC809 and RCC802) and Bathycoccus
AUTHORS: francois-yves bouget, Francois-Yves Bouget

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes the preparation of cells and introduction of DNA into the cells by electroporation.  For selection of stable transformants or measure of transient gene expression see related protocols.</div></div>

[STEPS]
?. [Cell preparation]
1) Starting from a culture of Ostreococcus tauri, RCC809 or Bathycoccus in stationary phase, innoculate cultures at 1 million cells/ml as determined by flow cytometry (Accuri C6 BD) in 200 ml plastic flasks in Artificial  Seawater supplemented with Keller medium supplement (trace metals, vitamins, nitrate and Phosphate as described in Djouani Tahri et al., PloS ONE 2011).For each transformation (including control), you should plan on using  50 ml de culture  in exponential phase. 2) Grow cells for 4 to 5 days depending on the light conditions, until they reach densities of  30 to 40.106 cells/ml.3) Count cells by flow cytometry. Check by SyBR Green II straining that bacterial contamination is below 2%. 4) Transfer lcultures to 50 ml Falcon tubes.5) Centrifuge at 8000g for 10 min at 4°C.6) Remove the supenatant, resuspend the cell  pellet in 1 ml de sorbitol 1M (pH 7.5) in H2O MQ, at 4°C. 7) Transférer the cell suspension to  1.5 ml eppendorf.8) Centrifuge at 8000g for 10 min at 4°C.9) Remove 900 µl of supernatant 10) Resuspend cells by gently pipeting.
?. [Electroporation of the transgene]
1) Add 5µg of transgene DNA  to cell suspension. Keep on ice for 5 minutes. The transgene consist of the high affinity phosphate promoter fused to the firefly luciferase (see Djouani Tahri et al., PloS one 2011).2) Transfer cells to a 2 mm electroporation cuvette (Biorad).3) Apply an electric field For  Ostreococcus tauri (OTTH595) : capacitance: 25µF, resistance 600 Ώ, voltage 1.35KV.For Ostreococcus sp RCC809 : capacitance: 25µF, resistance 600 Ώ, voltage 1.4KV.For Bathycoccus (RC4222) : capacitance: 25µF, resistance 600 Ώ, voltage 1.5KV.For Ostreococcus lucimarinus RCC802 : capacitance: 25µF, resistance 600 Ώ, voltage 1.2KV.4) Add 1ml of fresh culture Medium to resuspend the cells.5) Add 40 ml of culture medium and transfer to a culture flask. 6) Incubate at 20°C overnight in a light incubator.At this stage, transient transgene expression can me measured or stable transformants can be selected (see relevant protocols).
TITLE: Utilizing the Public GenomeTrakr Database for Foodborne Pathogen Traceback
AUTHORS: Ruth Timme, Maria Sanchez, Marc Allard

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol outlines the all the steps necessary to become a GenomeTrakr data contributor. GenomeTrakr is an international genomic reference database of mostly food and environmental isolates from foodborne pathogens. The data and analyses are housed at the National Center for Biotechnology Information (NCBI), which is a database freely available to anyone in the world. The Pathogen Detection browser at NCBI computes daily cluster results adding the newly submitted data to the existing phylogenetic clusters of closely related genomes. Contributors to this database can see how their new isolates are related to the real-time foodborne pathogen surveillance program established in the USA and a few other countries, and at the same time adding valuable new data to the reference database.</div><br/><div class = "text-block">------</div><br/><div class = "text-block">Although originally published as a Chapter in Methods and Protocols, Foodborne Bacterial Pathogens, the protocol has since been adapted and split into four separate protocols all of which are contained in this collection.</div></div>

[STEPS]
TITLE: PDMS Fabrication
AUTHORS: Kenneth Schackart, Kattika Kaarj

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol details how to make a small piece of polydimethylsiloxane (PDMS)</div></div>

[STEPS]
?. [Prepare surface]
Wipe the inside of the petri dish lid with 70% ethanol solution using a Kimwipe® to rid the surface of debris.
?. [Prepare surface]
Place petri dish lid(s) and a 10 cm × 10 cm piece of foil in the dessicator, in the fume hood.
?. [Prepare surface]
Add Tridecafluoro-1,1,2,2-tetrahydrooctyl trichlorosilane to the piece of aluminum foil.
2 µl
Chemical is highly corrosive, do not inhale or get on skin.
?. [Prepare surface]
Dessicate under vacuum for  to vaporize reagent.
Press down on the lid as you apply vacuum. Once vacuum is achieved the lid should be able to be lifted.
?. [Mix polymer]
Add PDMS curing agent and PDMS polymer to 2 mL tube.
100 µl
900 µl
?. [Mix polymer]
Mix the solution with an inoculation loop until there are small bubbles throughout the solution. This will take approximately .
?. [Degas polymer]
Pour the mixed PDMS into petri dish, and try to get all of the polymer out of the tube using a pipette tip or inoculation loop. Evenly spread the polymer in the petri dish by tilting.
?. [Degas polymer]
Dessicate under vacuum for .
?. [Cure polymer]
Place petri dish on hot plate at for covered in aluminum foil.
?. [Harvest polymer]
Carefully peel PDMS from petridish.
?. [Harvest polymer]
Using a blade, cut out a piece about 1 cm × 1 cm and set on glass microscope slide. This will be your PDMS surface for water contact angle analysis.
TITLE: (Epstein, Kim, Schanke) Trial K+ Minimal Media Recipe
AUTHORS: Elizabeth Fozo

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Trial K</span><span style = "vertical-align:super;">+</span><span> Minimal Media Recipe</span></div></div>

[STEPS]
?. [1L recipe]
Rinse the beaker with DI H2OFill to ~900mL MiliQ waterSet to mix, no heat.Choose between:In media room/dry goodsAbove lab benchesIn the fridgeFill to 1L with MiliQ and mix till dissolved (can be on low heat)Check pH (should be ~7), [K+] (normally ~8 ppm), and filter
?. [250mL recipe]
Rinse the beaker with DI H2OFill to ~200mL MiliQSet to mix, no heatChoose between:In media room/dry goodsAbove lab benchesIn the fridgeFill to 250mL with MiliQ and mix till dissolved (can be on low heat)Check pH (should be ~7), [K+] (normally ~8 ppm), and filter
?. [1L recipe]
In media room/dry goods6.5302g/L of Na2HPO43.16 g/L of NaH2PO40.52855 g/L of (NH4)2SO4500mg of amino acid mix 3
?. [1L recipe]
Above lab benchesMgSO4, 400uL of 1M stock - 0.4 mM5mL of 10mg/mL amino acid mix 1 - 50 mg/L5mL of 10mg/mL amino acid mix 2 - 50 mg/L Glycerol, 5mL of 80% stock    - 0.4%sodium citrate, 1mL of stock of 1M - 1 mM
?. [1L recipe]
In fridge0.1mL of 10mg/mL Thiamine -  I mg/LFeSO4, 600uLof stock of 10 mM - 6uM
?. [250mL recipe]
In media room/dry goods1.633g of Na2HPO40.79g of NaH2PO40.132g of (NH4)2SO4125mg of amino acid mix 3
?. [250mL recipe]
Above lab benchesMgSO4, 100uL of 1M stock - 0.4 mM1.25mL of 10mg/mL amino acid mix 1 - 50 mg/L1.25mL of 10mg/mL amino acid mix 2 - 50 mg/L Glycerol, 1.25mL of 80% stock    - 0.4%(glucose control, 5mL 20% stock)sodium citrate, 250uL of stock of 1M - 1 mM
?. [250mL recipe]
In fridge25uL of 10mg/mL Thiamine -  I mg/LFeSO4, 150uL of stock of 10 mM - 6uM
TITLE: Opomba protocol
AUTHORS: Matt H

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Hi there</span><a href="#" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;"> </span></a><span style = ":;"> </span></div></div>

[STEPS]
?. [Pre-work things]
Warm-upGet reagents from the  fridge
?. [Pre-work things]
?. [Pre-work things]
TITLE: Tau Thioflavin T Assay
AUTHORS: Alexandra Netter-Glangeaud

[STEPS]
?. A 1mM stock solution of Thioflavin T was prepared in dH2O (prepared fresh and filtered through a 0.2 µm syringe filter).
?. The thioflavin T was diluted in PBS pH 7.4 so that the final Thioflavin T concentration in each well was 25 µM (volume per well = 100 µL).
?. 10 uM Heparin was added to each well.
?. Tau aliquots were thawed at room temperature just before use.
?. Either fibril or monomer (or both) was added to the appropriate wells. The well contents were pipetted up and down to mix.
?. The plate was sealed and placed in a shaking incubator (800 rpm) at
?. Fluorescence was measured on a Molecular Devices Gemini XPS Microplate Reader using Softmax Pro software version 6.5.1. XPS Microplate Reader Settings:Temperature: 37°CRead Type: Well ScanWavelength: Excitation at 450 nm and Emission at 485 nmPMT Gain: AutomaticFlashes per read: 6Shake: 20 seconds before read
?. The plate was re-sealed and placed into the shaking incubator at 37°C.
?. Readings were taking at regular intervals from 1 hour to 72 hours.
TITLE: COVIRAP testing protocol
AUTHORS: Suman Chakraborty,  Arindam Mondal,  Aditya Bandopadhyay, Sujay Kumar Biswas,  Saptarshi Banerjee,  Nandita Kedia,  Subhamoy Chatterjee,  Aratrika de, Indranath Banerjee

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:justify">We have developed an RT-LAMP based protocol for isothermal amplification and subsequent colorimetric detection of the COVID19 infection. This protocol essentially relies upon the RT-LAMP mediated reverse transcription and subsequent amplification into large copies of cDNA products in the first step of the protocol. Subsequently, we have fused a second step where we label the cDNA with a fluorophore based probe to increase the sensitivity and specificity of the results. This step also makes the overall process to be compatible with the paper strip based detection technique where the product from the second step gets captured by the molecule impregnated in the paper strip. This leads to the development of the control and test line which could be detected with a smart phone based app for the prediction and rapid dissemination of the result. </div><style>
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[STEPS]
?. [DNA/RNA amplification]
Aliquot   RT-LAMP master mix into PCR tubes considering the total number of reactions. For one RNA sample, three reactions need to be carried out; one corresponding to RNaseP (internal control) and two corresponding to viral gene targets (N gene and ORF 1b).
10 µl
?. [DNA/RNA amplification]
Add specific primers (mixture of all the RT LAMP primers) corresponding to RNaseP, N gene and ORF 1b.
?. [DNA/RNA amplification]
Add  to   of RNA extracted from patient swab sample.
1 µl
9 µl
?. [DNA/RNA amplification]
Switch on the device. Put the microchamber on the isothermal heating platform of the portable device with the help of microchamber holder
?. [DNA/RNA amplification]
The target temperature for isothermal heating of the RNA mix is pre-set at  . This isothermal heating will continue for next  minutes. System will increase the temperature to  after 30 minutes and at  will continue for  minutes
65 °C
85 °C
85 °C
?. [DNA/RNA amplification]
*Add specific probes into the microchamber and continue the isothermal heating at specified temperature for a specific time.  Then cool down the chamber to room temperature at *This step is IP protected by this team.
25 °C
?. [Colorimetric Detection]
Apply the amplified reaction mixtures to the paper-strip and allow the lateral flow to occur. This will lead to the development of control and test lines on the strip.
?. [Colorimetric Detection]
Put the smartphone in its designated place. Wait for  to  minutes. and then start the smartphone app.
?. [Colorimetric Detection]
The result will be dispalyed onto the smartphone screen after 1-2 minutes.
TITLE: NucleoSpin® Gel and PCR Clean-up
AUTHORS: iGEM Dusseldorf

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><ul style = "list-style-type:disc;"><li style = "counter-reset:ol0;list-style-type:disc;">Buffer NTI</li><li style = "counter-reset:ol0;list-style-type:disc;">NucleoSpin® Gel and PCR Clean-up Column </li><li style = "counter-reset:ol0;list-style-type:disc;">NucleoSpin® Gel and PCR Clean-up Collection Tube (2ml)</li><li style = "counter-reset:ol0;list-style-type:disc;">Buffer NT3 </li><li style = "counter-reset:ol0;list-style-type:disc;">Buffer NE / nuclease-free water</li><li style = "counter-reset:ol0;list-style-type:disc;"></li></ul></div><div class = "text-block"><span style = "font-weight:bold;">Solubilize gel slice</span></div><div class = "text-block">For each 100 mg of agarose gel < 2 % add 200 μL Buffer NTI. For gels containing > 2 % agarose, double the volume of Buffer NTI. Incubate sample for 5–10 min at 50 °C. Vortex the sample briefly every 2–3 min until the gel slice is completely dissolved! </div><div class = "text-block"><span style = "font-weight:bold;">Bind DNA</span></div><div class = "text-block">Place a NucleoSpin® Gel and PCR Clean-up Column into a Collection Tube (2 mL) and load up to 700 μL sample. Centrifuge for 30 s at 11,000 x g. Discard flow-through and place the column back into the collection tube. Load remaining sample if necessary and repeat the centrifugation step. </div><div class = "text-block"><span style = "font-weight:bold;">Wash silica membrane </span></div><div class = "text-block">Add 700 μL Buffer NT3 to the NucleoSpin® Gel  and PCR Clean-up Column. Centrifuge for 30 s at 11,000 x g. Discard flow-through and place the column back into the collection tube. </div><div class = "text-block"><span style = "font-weight:bold;">Dry silica membrane </span></div><div class = "text-block">Centrifuge for 1 min at 11,000 x g to remove Buffer NT3 completely. Make sure the spin column does not come in contact with the flow-through while removing it from the centrifuge and the collection tube. Note: Residual ethanol from Buffer NT3 might inhibit enzymatic reactions. Total removal of ethanol can be achieved by incubating the columns for 2–5 min at 70 °C prior to elution. </div><div class = "text-block"><span style = "font-weight:bold;">Elute DNA</span></div><div class = "text-block">Place the NucleoSpin® Gel and PCR Clean-up Column into a new 1.5 mL microcentrifuge tube (not provided). Add 15–30 μL Buffer NE and incubate at room temperature (18–25 °C) for 1 min. Centrifuge for 1 min at 11,000 x g.</div></div>

[STEPS]
TITLE: ACTION Biomarker Study
AUTHORS: Fiona A. Hagenbeek, Jenny van Dongen, Peter J. Roetman, Erik A. Ehli, Meike Bartels, Robert R. J. M. Vermeiren, Dorret I. Boomsma, ACTION Consortium

[DESCRIPTION]
The goal of ACTION project (Aggression in Children: Unraveling gene-environment interplay to inform Treatment and InterventiON strategies) was to identify precursors, features, and consequences of childhood aggression using epidemiological and omics-driven studies. A considerable effort within ACTION was the collection of new omics data in children with low and high aggressive behavior. To this end, the ACTION project included large-scale collection of biological samples in children from the Netherlands Twin Register (NTR), a twin cohort from the general population, and a cohort of children referred for psychiatric treatment (LUMC-Curium). Together these cohorts comprise the ACTION Biomarker Study. This documentation describes the study design and data collection for the ACTION Biomarker Study.

[STEPS]
TITLE: Protein Aggregation Capture
AUTHORS: Mark Kinnin

[DESCRIPTION]
PAC Adapted from Batth et al., 2019

[STEPS]
SECTION: Equilibration
1. Add 5 µL of   to 1.5mL LoBind Eppendorf tube



SECTION: Equilibration
1.1. Place tube on magnet and allow Microparticles to clear 10 s 

SECTION: Equilibration
1.2. Remove storage solution and discard
SECTION: Equilibration
2. Equilibrate Microparticles by adding  100 µL   and mix by gentle agitation

SECTION: Equilibration
2.1. Place tube on magnet and allow Microparticles to clear 10 s 

SECTION: Equilibration
2.2. Remove supernatant and discard
SECTION: Equilibration
3. Add 100 µL    and mix by gentle agitation

SECTION: Equilibration
3.1. Place tube on magnet and allow Microparticles to clear 10 s 
SECTION: Equilibration
3.2. Remove supernatant and discard
SECTION: Equilibration
4. Add 2 µL sample in triplicate

SECTION: Equilibration
5. Add   to a concentration of 70% and mix once by pipette to create a uniform suspension

SECTION: Equilibration
6. Allow to rest 10 min 

SECTION: Wash - DO NOT REMOVE FROM MAGNET
7. Place tube on magnet and allow Microparticles to clear 10 s 

SECTION: Wash - DO NOT REMOVE FROM MAGNET
7.1. Remove supernatant and discard
SECTION: Wash - DO NOT REMOVE FROM MAGNET
8. Add 1 mL   and incubate for 10 s 

SECTION: Wash - DO NOT REMOVE FROM MAGNET
9. Add 1 mL   and incubate for 10 s 

SECTION: Wash - DO NOT REMOVE FROM MAGNET
8.1. Remove supernatant
SECTION: Wash - DO NOT REMOVE FROM MAGNET
9.1. Remove supernatant
SECTION: Digestion
11. Add 5 µg   and incubate for 240 min 
SECTION: Digestion
10. Remove tube from magnet and add pH 8.5   and mix throughly
SECTION: Digestion
12. Quench to a final concentration 1 % volume   

SECTION: Digestion
13. Mix and place samples on magnet for 1 min and transfer supernatant to tubes

SECTION: Digestion
14. Wash beads with 50-100 µL 1 % volume   for 2 min with continous mixing

SECTION: Digestion
15. Centrifuge tubes 20000 x g, 10 min 

SECTION: Digestion
14.1. Remove supernatant and pool with eluate from Step 13
SECTION: Digestion
16. Transfer supernatant to new tube and freeze down at -80 °C
TITLE: Assembly, Annotation, Quantification, and Differential Expression Analysis of Shorea sp. Transcriptome
AUTHORS: Ahmad Husaini AHS Suhaimi

[DESCRIPTION]
Assembly, annotation, and quantification of transcripts from RNA-seq reads of Shorea sp. transcriptome followed by differential expression analysis using open source tools.

List of tools:
Trinity (Home · trinityrnaseq/trinityrnaseq Wiki · GitHub)
TransDecoder (Home · TransDecoder/TransDecoder Wiki · GitHub)
NCBI Blast suite (Download BLAST Software and Databases Documentation (nih.gov))
InterProScan (Download - InterPro (ebi.ac.uk))
CDHIT suite (CD-HIT Official Website (ucsd.edu))
Salmon (Salmon - Salmon 1.6.0 documentation)
DESeq2 (Bioconductor - DESeq2)

[STEPS]
SECTION: Transcript Assembly
2. 
Assemble the reads using Trinity assembler
 


SECTION: Transcript Assembly
1. Obtain the assembly statistics
 

SECTION: Transcript Annotation
7. Query the nonredundant nucleotide sequences against A. thaliana proteome
 

SECTION: Transcript Annotation
8. Query the nonredundant protein sequences against Pfam, PANTHER, GO, and KEGG databases
 

SECTION: Transcript Quantification
9. Align and estimate transcript abundance using Salmon
 

SECTION: Transcript Quantification
10. Construct matrices of counts and normalized values
 

SECTION: Differential Expression Analysis
11. Perform pairwise comparison between the time points
 

SECTION: Differential Expression Analysis
12. Extract the differentially expressed transcripts at log2 fold change ≥ 1, FDR < 0.05
 

SECTION: Removal of Transcript Redundancy
3. Extract the longest open reading frame (ORF) using TransDecoder
 

SECTION: Removal of Transcript Redundancy
4. Query the extracted ORFs against Uniprot database using blastp
 

SECTION: Removal of Transcript Redundancy
5. Predict the single best ORF for each transcript
 

SECTION: Removal of Transcript Redundancy
6. Remove highly similar protein sequences using CD-HIT
TITLE: Abeoforma whisleri_culture method
AUTHORS: Meritxell  Antó

[STEPS]
?. [Growing medium]
BD Difco Marine Broth 2216 • Marine Agar 2216 https://legacy.bd.com/europe/regulatory/Assets/IFU/Difco_BBL/212185.pdf
?. [Growth conditions]
Temperature 17ºC
?. [Cryopreservation and recovery (from liquid cultures)]
Cryopreservation  (10%DMSO in 1M sorbitol or 10%(glycerol 60%) in sorbitol 1M)1. Pre-grow the culture in appropriate growing medium until almost saturated but not too old.2. Mix 1.35mL of the culture with 150 ul of DMSO in 1M sorbitol or (glycerol 60%) in sorbitol 1M in the screw-top cryovials.3. Transfer the cryovials to the Freezing container, Nalgene®Mr. Frosty unit. Place it in the -80ºC freezer and leave it overnight.4. Remove cryovials from the Freezing container and store in appropriate boxes for -80ºC.Recovery1. Fill a flask with 10mL fresh Growing medium.2. Take out the frozen stock from the freezer, carry it to the culture hood in a cryo box to prevent it from thawing.3. With a pipette tip (or a loop), take a chunk of the frozen stock and resuspend it in the culture flask (~10uL is probably sufficient). Take the frozen stock back to the -80ºC as rapidly as possible.4. Let it recover for at least 3-4 days and proceed with another transfer in a fresh medium even if the cells are not completely grown it will help to dilute the remaining DMSO from the cryopreservation, the time will vary depending on the amount of the initial inoculum and the culture type.*NOTE: because of DMSO some precipitates may appear in the culture.All protists must be stored in liquid nitrogen for long-term storage. Although storage at -80°C is sometimes sufficient for a temporary alternative.
TITLE: GUV preparation  and  assay
AUTHORS: Chunmei  Chang

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">LC3 lipidation on GUVs</div></div>

[STEPS]
?. GUV Preparation
?. Clean the coverslips of 25 mm diameter.
?. Coat cleaned coverslips with 60 μL 5% (w/w) polyvinyl alcohol (PVA) with a molecular weight of 145,000 (Millipore).
?. Place the coated coverslip in a heating incubator at 60 °C to dry the PVA film for 30 min.
?. Spread a lipid mixture with a molar composition of 64.8% DOPC, 20% DOPE, 10% POPI, 5% DOPS and 0.2% Atto647N DOPE at 1 mg/ml uniformly onto the PVA film.
?. Put the lipid-coated coverslip under vacuum overnight to evaporate the solvent.
?. Use 400 μL 400 mOsm sucrose solution for swelling for 1 h at room temperature
?. Harvest the GUVs and use them with 12 h.
?. Acquire time-lapse images  in multitracking mode on a Nikon A1 confocal microscope.
?. Pick random views for imaging within 5 min.
?. Add 10 µL GUVs to initiate the reaction.
?. Make a 120 µL reaction mixtures with the proteins and 50 μM ATP. The final concentration the each protein is 5 µM GST-4xUb, 500 nM cargo receptors, 25 nM ULK1 complex, 25 nM PI3KC3-C1 complex, 100 nM WIPI2d, 50 nM ATG12–ATG5-ATG16 complex, 100 nM ATG7, 100 nM ATG3, and 500 nM mCherry-LC3B.
?. Wash the coated chamber three times with reaction buffer (20 mM HEPES at pH 8.0, 190 mM NaCl and 1 mM TCEP).
?. Coat the chamber with 5 mg/ml β casein for 30 min.
?. Set up the reaction in an eight-well observation chamber (Lab Tek) at room temperature.
?. GUV Assay