eamag/protocols-with-steps huggingface dataset:
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>We simulate paired-end reads for testing the accuracy and sensitivity of our computer program for detection of transportable element (TE) insertions (also called Mobile Element Insertions, MEIs). we named the software "Specific Insertions Detector (SID)".</p>

[BEFORE_START]
<p>1.Setup PIRS software and SID; 2. Download the sequences of transportable elements (FASTA file)  and human genome assembly sequence (hg19, FASTA file).</p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?.
TITLE: Calculating multiplicity of infection (MOI)
AUTHORS: Januka  Athukoralage, Adair  Borges

[DESCRIPTION]
This is a simple protocol for the calculations that we routinely do to determine phage multiplicity of infection (MOI) in our experiments.

It's really important to know what MOI you are using. Ideally, you would perform these quantifications before your experiments such that you have a good ballpark understanding of what your multiplicity of infection is in your experiment. This also allows you to prepare phage dilutions to more precisely hit your target MOI.

We will also sometimes plate out bacteria and phage day-of using our experimental samples to give us an exact measurement of our MOI based on the culture that we used and to double-check that our phage dilution was correct. 

[STEPS]
SECTION: Calculating colony-forming units per mL (CFU/mL)
1. Grow your bacteria to your target OD600 using the same culture conditions that you will use in your experiments.
SECTION: Calculating colony-forming units per mL (CFU/mL)
2. Create a 10-fold dilution series of your bacterial culture. Use glass beads to seed an LB agar plate with 100 µl of the 10-4, 10-5 , 10-6, and 10-7 dilutions of each subculture and incubate overnight. These dilutions typically yield a suitable number of colonies for accurate counting by eye. If they do not, you will need to adjust up or down depending on the exact details of what you are doing.

Count colonies the next day, and calculate how many colony-forming units (CFUs) are present in 1 mL of culture.

CFU/mL = (# colonies × dilution factor) ÷ 0.1 mL 
2. 
SECTION: Calculating plaque-forming units per mL (PFU/mL)
3. Infect 150 µL bacterial culture with 10 µL phage, respectively, from 10-fold dilution series of phage stocks. Aim for dilutions that will give you single countable plaques. Mix with with 3 mL of 0.5% (0.5 g/100 mL) agarose dissolved in LB supplemented with 1 mM MgSO4, pour onto LB plates, and incubate at 37 °C overnight.

Count plaques the next day, and calculate how many plaque-forming units (PFUs) are present in 1 mL of phage stock.

PFU/mL = (# plaques × dilution factor) ÷ 0.01 mL 
SECTION: Calculating multiplicity of infection (MOI)
4. In our experiments, we generally aim for an MOI of ~10 to ensure that the majority of cells are infected with phage. However, you will need to determine the MOI appropriate for your experiments based on the biological phenomena you are studying. We also aim to have the phage volume be <= 10% of the bacterial culture volume. So, we might use 500 µl of bacterial culture and 50 µl of phage stock. Again, you will need to determine which volumes are appropriate for your experiment. Here is how we calculate MOI using PFU/mL and CFU/mL.

PFU/mL × phage volume in mL = Total PFU

CFU/mL × bacterial volume in mL  = Total CFU

Total PFU ÷ Total CFU = MOI
TITLE: In vitro phosphatase assay
AUTHORS: Elias Adriaenssens

[DESCRIPTION]
This protocol describes in vitro phosphatase assay.

[STEPS]
SECTION: In vitro phosphatase assay
1. Seed HAP1 wild-type or FIP200 knockout cells in 6 well plates and grow until confluency.
SECTION: In vitro phosphatase assay
2. Collect cells by trypsinization and pellet by centrifugation at 300 x g, 5 min, 4 °C.

SECTION: In vitro phosphatase assay
3. After a PBS wash to remove the remaining cell medium, resuspend the cell pellets in lysis buffer.
SECTION: In vitro phosphatase assay
4. Lyse the samples for 20 min on ice. Clear the cell lysates by centrifugation at 20000 x g, 10 min, 4 °C.

SECTION: In vitro phosphatase assay
5. Determine the protein concentrations of the cleared protein lysates with the Pierce Detergent Compatible Bradford Assay Kit (23246, Thermo Fisher).
SECTION: In vitro phosphatase assay
6. For both samples, wild-type and FIP200 knockout lysates, incubate 100 µg of cell lysate with 5 µL of 10x NEBuffer for Protein MetalloPhosphatases (P0753, New England Biolabs) and 5 µL of 10 millimolar (mM) of MnCl2 to make a total reaction volume of 50 µL.
SECTION: In vitro phosphatase assay
7. Add 1 µL of Lambda Protein Phosphatase (NEB) to the reaction and incubate the samples at 30 °C for the indicated time.
SECTION: In vitro phosphatase assay
8. Terminate the phosphatase reactions by the addition of 6x Protein Loading dye and heat inactivation at 95 °C for 5 min. 
SECTION: In vitro phosphatase assay
9. Analyze the samples by western blot analysis as described above.
TITLE: VPS13D DNA plasmid generation
AUTHORS: Andrés Guillén-Samander, Marianna Leonzino, Pietro De Camilli

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>        This protocol describes the basic molecular cloning technique utilized for the generation of VPS13D constructs in </span><a href="https://doi.org/10.1083/jcb.202010004" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://doi.org/10.1083/jcb.202010004</span></a><span>. This protocol and the enzymes included in it are commercialized by Takara Bio.</span></div><br/><div class = "text-block"><span>        Due to low expression of big DNA constructs (>10kb), we decided to generate a codon-optimized cDNA encoding for human VPS13D, including an mScarlet fluorescent protein after aminoacid 1576 flanked by BamHI restriction enzyme sites. The construct was generated by and purchased from Genscript. From this initial construct, we generated several VPS13D constructs. The specific primers and enzymes used for each construct are included in Table 1 of our manuscript: </span><a href="https://doi.org/10.1083/jcb.202010004" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://doi.org/10.1083/jcb.202010004</span></a><span>" . For most of our cloning procedures, Infusion cloning (Takara) was used.</span></div></div>

[STEPS]
?. [VPS13D DNA plasmid generation]
Linearize the backbone with restriction enzymes or by PCR amplification.
?. [VPS13D DNA plasmid generation]
Amplify the insert by PCR with primers including 15nt of overlapping sequence with the target backbone.
Suggestion: Takara has an online tool to help with primer design at: https://go.shr.lc/3daJf0E
?. [VPS13D DNA plasmid generation]
Run both products in an agarose gel to confirm expected size and purify the DNA from gel using a NucleoSpin Gel and PCR Clean-up kit (Takara).
?. [VPS13D DNA plasmid generation]
Ligate the linearized backbone and the insert with the Infusion enzyme mix (Takara).
Suggestion: We found that big DNA constructs accumulate mutations easily when amplified in bacteria, this can be minimized by growing bacteria at  .
TITLE: Preparation of electrocompetent Escherichia coli
AUTHORS: lewis.bingle 

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Preparation of competent </span><span style = "font-style:italic;">Escherichia coli</span><span> cells for electroporation.</span></div></div>

[STEPS]
?. Inoculate starter culture of the desired strain from single colony (e.g. LB broth in universal bottle). Incubate (37°C with shaking at 200-220 rpm) .
5 mL
?. Dilute overnight culture 1 / 100 into  fresh LB broth (supplemented with appropriate antibiotic if required) and incubate (37°C with shaking at 200-220 rpm) to mid-log phase growth (OD600nm of between 0.5 and 0.7).
25 mL
?. Decant bacteria into 50 ml Falcon (centrifuge) tube and chill on ice (approx. ).
?. Centrifuge   for   (use refrigerated centrifuge if possible, or pre-chill centrifuge rotor / bucket).* Discard supernatant.
Centrifuge: 4000 34, 4 10
?. Wash the cells 3x with ice-cold, sterile aqueous glycerol solution   (can use sterile distilled water) in decreasing volumes of ,  and  (centrifuge as in step 4 between each wash).
25 mL
12 mL
6 mL
?. Resuspend final cell pellet in  of sterile 10% (w/v) glycerol (or water)  .
300 µl
on ice
?. Store electrocompetent cells on ice for immediate use or snap-freeze in liquid nitrogen for long-term storage at .
-80 °C
TITLE: pH Testing of Coffee Samples
AUTHORS: Shannon.logan

[DESCRIPTION]
Measure the pH levels of coffee samples across a range of roasts. 

[STEPS]
2. Boil kettle with 250ml of deionised water.
1. Grind coffee to desired consistency and weigh 20g to put in cafetiere. 
3. Pour boiling water into beaker and allow to cool to 93°C, using a digital thermometer to ensure accuracy of temperature.
4. Once the water has cooled to the desired temperature, pour into the cafetiere and allow to brew for 4 minutes, using a timer/stopclock. 
5. Once the coffee has brewed, use the cafetiere as stated in the instruction manual, and pour coffee into beaker. Test the coffee with the pH meter. 

6. Repeat this process with all coffee samples.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
This protocol was developed for the human breast cell atlas (HBCA) project to obtain high-viability cell suspensions from freshly dissociated breast tissues from human patients. There are two options for performing this protocol: rapid-dissociation (15-30 min) or exhaustive dissociation (overnight). In our experience the rapid dissociation protocol will provide higher cell viability and better representation of breast cell types, however the total number of cells dissociated may be lower after enumeration. In contrast the exhaustive protocol provides higher cell numbers and maintains good cell viability but may result in skewing of cell type representations over the long time period. The rapid-dissociation protocol was used to generate most of the cell suspensions for the MD Anderson human breast cell atlas project and has been optimized and tested more extensively.

[STEPS]
SECTION: Making 10xcollagenase solution
?. 
SECTION: Dissociation solution (DS)
?. 
SECTION: Mincing
?. 
SECTION: Mincing
?. 
SECTION: Dissociation
?. 
SECTION: Dissociation
?. 
SECTION: Dissociation
?. 
SECTION: Spin
?. 
SECTION: Spin
?. 
SECTION: Resuspending
?. 
SECTION: Resuspending
?. 
SECTION: Centrifugation
?. 
SECTION: RBC lysis
?. 
SECTION: RBC lysis
?. 
SECTION: Centrifugation
?. 
SECTION: Centrifugation
?. 
SECTION: Centrifugation
?. 
SECTION: Centrifugation
?. 
SECTION: Wash
?. 
SECTION: Wash
?. 
SECTION: Wash
?. 
SECTION: For 10X genomics RNA experiments
?. 
SECTION: For 10X genomics RNA experiments
?. 
SECTION: For 10X genomics RNA experiments
?. 
SECTION: Dissociation
?. 
SECTION: Dissociation
?. 
SECTION: Spin
?. 
SECTION: Spin
?. 
SECTION: Resuspending
?. 
SECTION: Resuspending
?. 
SECTION: Centrifugation
?. 
SECTION: RBC lysis
?. 
SECTION: RBC lysis
?. 
SECTION: Centrifugation
?. 
SECTION: Centrifugation
?. 
SECTION: Centrifugation
?. 
SECTION: Centrifugation
?. 
SECTION: Wash
?. 
SECTION: Wash
?. 
SECTION: Wash
?. 
SECTION: For 10X genomics RNA experiments
?. 
SECTION: For 10X genomics RNA experiments
?. 
SECTION: For 10X genomics RNA experiments
?.
TITLE: Rehabilitation protocol after varization osteotomy - Case Study
AUTHORS: Sebastião Santos

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Background: </span><span>Knee rehabilitation using therapeutic exercises in the aquatic environment has been used in order to initiate lower limb rehabilitation earlier and improve joint movements, as it reduces pain and allows for earlier weight bearing. Objective: To develop and describe the effects of an aquatic therapeutic exercise program in a case study of tibial osteotomy and varus femur. </span><span style = "font-weight:bold;">Methods</span><span>: A 15-year-old girl undergoing a tibial and femoral varus osteotomy completed an aquatic therapeutic exercise program consisting of a total of 30 sessions (3 sessions per week of 60 minutes). The subject was evaluated at two different times (at the beginning and end of the intervention). </span><span style = "font-weight:bold;">Results</span><span>: There was an improvement in the various parameters evaluated (knee flexion: 66º vs 125º; knee extension: -7º vs -1º; pain on movement: 6/10 vs 0/10). </span><span style = "font-weight:bold;">Conclusion</span><span>: The aquatic therapeutic exercise program used in this study has beneficial effects in a clinical case of tibial osteotomy and varus femur.</span></div></div>

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>SNPs calling protocol for analysis the genetic polymorphism among natural color variants. This protocol accompanies the following <em>GigaScience</em> publication:</p>
<p> </p>
<p>Jihoon Jo, et al.  (2016): Draft genome of the sea cucumber Apostichopus japonicus and genetic polymorphism among color variants. <em>GigaScience</em>...</p>

[STEPS]
?. 
?. 
?. 
?. 
?.
TITLE: DNA extraction (BOMB_Soil)
AUTHORS: Tsu-Chun  Hung, Yin-Tse    Huang, Hsin-Mao   Wu

[DESCRIPTION]
DNA extraction (BOMB_Soil)

[STEPS]
SECTION: Sample Collection
2. Add 200 µL of 1mm beads to 2ml enppendorf tube

SECTION: Sample Collection
3. Add200 µL of 0.5mm beads to 2ml enppendorf tube

SECTION: Sample Collection
4. Add 225 µL of TE buffer to 2ml enppendorf tube
 

SECTION: Sample Collection
5. Add 375 µL of lysis buffer to 2ml enppendorf tube
 


SECTION: Sample Collection
6. Take 2ml enppendorf tube out of the laminar flow and transfer soil samples to the 2ml enppendorf tube
SECTION: Sample crush
7. Put 2ml eppendorf tube in mixmill for sample crush, at this condition: 30 rpm/s, for 4mins 4 min 
SECTION: Centrifugation
8. Put 2ml eppendorf tube in centrifuge for centrifugation, at this condition:17.0 x g, 3 min, 25 °C 
SECTION: DNA purification
17. Add 350 µL of isopropanol to the 1st well of 96 well plate

   

SECTION: DNA purification
17.1. Add 125 µL of magnetic beads (10 mg/ml) to the 1st well of 96 deep well plate 

 


SECTION: DNA purification
17.2. Add 200 µL of the sample (lysate) from the 2ml centrifuged tube to the 1st well of 96 deep well plate 
 

SECTION: DNA purification
18. Add 400 µL of isopropanol to the 2nd well of 96 deep well plate 
SECTION: DNA purification
19. Add 300 µL of 80% enthanol to the  3rd well of 96 deep well plate 
SECTION: DNA purification
20. Add 300 µL of 80% enthanol to the 4th well of 96 deep well plate 
SECTION: DNA purification
21. Add 100 µL of DD water to the 5th well of 96 deep well plate 
SECTION: DNA purification
22. Put the prepared 96 deep well plate in the automated DNA extraction machine

SECTION: DNA purification
23. After the extraction is done, collect 100 µL of the eluted sample as the DNA template for downstream experiments

SECTION: Sample Collection
1. Measuring 250 mg  for a soil sample

SECTION: Remove proteins and humic acid
9. Add 250 µL  0.133M Ammonium acetate in a new 2ml enppendorf tube

SECTION: Remove proteins and humic acid
10. Transfer supernatant 300 µL  of step 8 into 2ml enppendorf tube in step 9

SECTION: Remove proteins and humic acid
11. Incubate on ice for 10 min 

SECTION: Remove proteins and humic acid
12. Put 2ml eppendorf tube in centrifuge for centrifugation, at this condition:17.0 x g, 3 min, 25 °C 
SECTION: Remove proteins and humic acid
13. Add 200 µL  0.06M Aluminium sulfate in another 2ml enppendorf tube

SECTION: Remove proteins and humic acid
14. Transfer step 12 supernatant 500 µL  in step 13’s 2ml enppendorf tube

SECTION: Remove proteins and humic acid
15. Incubate on ice for 10 min 

SECTION: Remove proteins and humic acid
16. Put 2ml eppendorf tube in centrifuge for centrifugation, at this condition:17.0 x g, 10 min, 25 °C
TITLE: Quantifying how FAIR is Hong Kong: The Hong Kong Shareability of Hong Kong University Research Experiment
AUTHORS: Scott Edmunds, Jesse Xiao, Hongling Zhou

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Using 59 HKU students undertaking the data curation module of the Master of Science in Library and Information Management (MLIM) course taught by some of the </span><span style = "font-style:italic;">GigaScience</span><span> team, a crowdsourced literature curation exercise was carried out to assess the data availability of HKU's papers published in </span><span style = "font-style:italic;">PLOS One</span><span> and </span><span style = "font-style:italic;">Scientific Reports</span><span> journals between 2016-2018. This experiment can be continued at other institutions, and has been presented to allow a teacher of a similar course to set this up for their own students.</span></div><div class = "text-block">This collection includes protocols for teachers trying to set up this exercise at a different university, and the protocol for the individual students carrying it out. Alongside a spreadsheet to input the results.</div></div>

[STEPS]
TITLE: WIPI2d Coprecipitation Assay
AUTHORS: lmstrong 

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Pulldown assay of WIPI2d</div></div>

[STEPS]
?. Equilibrate 10uL of GST resin to pulldown buffer. To do this, pipette 40uL of slurry (so 20uL of resin) into a 1.7uL eppy. Add >500mL of wash buffer. Slow spin to pellet resin. ~1000rpm for 1 minute should be good. Repeat X3
?. Add 10 μM purified WIPI2d and either with 20 μM of GST or GST-ATG16L1(207-230). Add buffer (25mM HEPES pH 7.4, 150mM NaCl, 1mM TCEP) to final volume of 150uL.
?. Let rock at 4C for 2 hour. Wash x3
?. Add 10uL of SDS-Loading dye to beads
?. Run beads on SDS-PAGE gel and stain with Coomassie.
TITLE:        ELISA for quantification of monocyte chemoattractant protein-1 (MCP-1/CCL2) in human serum or plasma
AUTHORS: Angel  Justiz-Vaillant, Belkis Ferrer-Cosme

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>The monocyte chemoattractant protein-</span><span style = "font-style:italic;">1</span><span style = "font-style:italic;font-style:italic;">(</span><span style = "font-style:italic;">MCP</span><span style = "font-style:italic;font-style:italic;">-</span><span style = "font-style:italic;">1</span><span>/CCL2) is a member of the C-C chemokine family, and it is a potent chemotactic factor for monocytes.</span></div></div>

[STEPS]
?. An anti-human monocyte chemoattractant protein-1 (MCP-1/CCL2) coating antibody is adsorbed onto the microwells by incubation overnight at 4°C with carbonate-bicarbonate buffer.
?. Add 50 µl of human serum or plasma into the wells. Human MCP-1/CCL2 present in the serum sample binds to antibodies adsorbed into the microwells.
?. The microplate is blocked with 3% non-fat milk-PBS buffer and later wash to remove unbound proteins.
?. Fifty (50) µl of biotin-conjugated anti-monocyte chemoattractant protein-1 (MCP-1/CCL2) antibody is added. The optimal dilution must be investigated.
?. The microplate is rewashed with PBS-Tween 20 buffer, pH 7.4.
?. One hundred µl of streptavidin-HRP conjugate is added and it binds to the biotin-conjugated anti-human MCP-1/CCL2 antibody.
?. The plate is washed following incubation to remove the unbound Streptavidin-HRP conjugate.
?. Add 100 µl of 3,3',5,5'- tetramethylbenzidine (TMB; Sigma-Aldrich) into each well.
?. Incubate the microwells in the dark for 15 min.
?. A colored product is formed in proportion to the quantity of MCP-1/CCL2 present in the sample or standard.
?. The reaction is terminated by addition of 100 µl 3M H2SO4  and the absorbance is measured at 450 nm.
?. A standard curve is made from 7 human MCP-1/CCL2 standard dilutions and the human MCP-1/CCL2 sample concentration is determined.
?. For better results place the microplate on a microplate shaker in every incubation.
TITLE: Biochemical assays and evaluation
AUTHORS: lparra

[DESCRIPTION]
Preparation of brain and Neuro2A lysates















[STEPS]
SECTION: Preparation of Brain and Neuro2A Lysates
1. 
Whole mouse brains were homogenized with a Dounce tissue grinder (20
times) in neuronal protein extraction reagent (N-PER) (Thermo Scientific)
containing protease/phosphatase inhibitors (Cell Signaling #5872).



SECTION: Preparation of Brain and Neuro2A Lysates
2. 
Triton X-100 (Sigma #X100-100ML) was added at
a final concentration of 1%, and the samples were incubated with rotation for 1
hour at 4 °C. 



SECTION: Preparation of Brain and Neuro2A Lysates
3. 
Samples were centrifuged at 10,000 × g
for 10 min at 4 °C, and the supernatant was collected.



SECTION: Preparation of Brain and Neuro2A Lysates
4. 
For Neuro2A lysates, cells were washed with 1X PBS 3 times and incubated
for 5 min on ice in the presence of N-PER reagent supplemented with protease
inhibitors.



SECTION: Preparation of Brain and Neuro2A Lysates
5. 
Samples were centrifuged at 10,000 × g
for 10 min at 4 °C to remove cellular debris. 



SECTION: Preparation of Brain and Neuro2A Lysates
6. 



Total protein
concentration was measured in brain and Neuro2A lysates (DC‱ Protein Assay Kit II, Biorad), and samples were
used in subsequent experiments.
TITLE: How to book a sorting slot in PPMS
AUTHORS: Daniel Gimenes

[DESCRIPTION]
Step by step protocol on how to book a cell sorter at EMBL's Flow Cytometry Core Facility

[STEPS]
SECTION: Access the PPMS calendar
1. Go to https://ppms.embl.de
SECTION: Access the PPMS calendar
2. Click on Flow Cytometry Core Facility

 

SECTION: Access the PPMS calendar
3. Login into your account

 

SECTION: Access the PPMS calendar
4. On the upper tabs, click on "BOOK"

 

SECTION: Access the PPMS calendar
5. Select one of the cell sorters that you want to book a slot

 

SECTION: Access the PPMS calendar
6. Use the options to move between weeks to find the desired date for your sorting

 


SECTION: Access the PPMS calendar
6.1. The calendar is colour coded:


 

Dark grey: Not available for booking
You will find this colour:
If you are not a novice (not a trained user), the over working and weekend hours slots will not be available for booking
If you are trying to book the instrument with less than 48 hours until the time of the appointment.

Light blue: Intervention (special configuration or not available)
This one highlights periods in which the instrument is blocked for scheduled interventions (such as a sorter bi-weekly aseptic cleaning) or for when the staff is not available to support the sorting (in such occasions, trained users might still have the opportunity to sort - please let us know).

*Notice that the instrument has a special configuration for the staff lunch period from 12:00-13:00 and it is also the period in which the staff has more time to change the nozzle between sessions. If you are booking an assisted sorting, please don't select these time slots!!!

Teal: Booked session that still has to be confirmed
Blue: Staff confirmed the session
Green: Staff confirmed the session for Operator Free (trained) users
Purple border: Session contains a comment to convey a special environment (such as 384 wp sorting)
Light grey: available slots for booking
SECTION: Access the PPMS calendar
6.3. A form will be shown for you to fill the information regarding your sorting. Once you are done, click on "Complete Booking"

 


SECTION: Access the PPMS calendar
6.2. To book a slot:

Select time slot by checking the 30 minutes segments boxes in the calendar
(Optional) If you are trained, check the "Operator Free" Box. If you want to add a comment to your booking check the "Comment" box.
Click on Book the selected sessions
 



SECTION: Access the PPMS calendar
6.4. Your newly booked slot should appear in the calendar!
The facility staff will then check and either confirm it directly, or if any issues arise you will be contacted for changes.