eamag/protocols-with-steps huggingface dataset:
TITLE: High Molecular Weight DNA extraction from tunicates 
AUTHORS: Marta   Wawrzyniak, Simon  Blanchoud

[DESCRIPTION]
This protocol has been successfully used with Botrylloides diegensis and has been based on the following publication (with small changes): 

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1016/0014-5793(89)80446-6


[GUIDELINES]
from step 8 on, avoid vortexing or vigorous shaking; invert the tube gently to avoid breaking the DNA.  

[STEPS]
1. Clean the slide from which you will take the colony of your interest. See Cleaning colonial ascidians.
2. Isolate a cleaned colony composed of approx. 20-30 zooids. 
5. Incubate at 55 °C for 10 min .




6. Add 4 µL of Proteinase K and mix by vortexing. 

2.1. Transfer to a 2 mL tube and spin at maximum speed for 2 min .
3. Homogenize the sample in500 µL  of TE buffer using a sterile plastic pestle. 
2.2. Remove the excess water. 
7. Incubate at 55 °C for 15 min .
8. Add 1 mL of phenol and mix well by inverting the tube until the phases are completely mixed.

9. Spin at 10000 rcf  for 5 min and carefully transfer the 500 µL  of the upper phase to a new 2 mL tube. 


10. Add 500 µL  of (1:1 v/v) phenol/chloroform and mix well by inverting the tube.


11. Spin at 10000 rcf  for 5 min and carefully collect the 300 µL  of upper phase to a new 2 mL tube. 

12. Precipitate the DNA by addition of 30 µL  3 M sodium acetate and 600 µL  of ethanol. Mix gently by inverting the tube.

13. Spin at 10000 rcf  for 3 min to pellet nucleic acids and carefully remove and discard supernatant.

14. Wash in 1 mL cold ethanol (-20 °C ) and invert gently several times.

16. Carefully remove and discard supernatant and place the tube up-side-down on a paper towel for 10 min .

17. Resuspend the pellet gently in RNase-free water at 37 °C for 60 min .

4. Add 500 µL of SDS and 4 µL of RNase and mix by vortexing. 

18. Quantify the DNA concentration and quality.
19. Store at -20 °C  or -80 °C (for longer storage).

15. Spin at 10000 rcf  for 2 min .
TITLE: SPM Addition in cells
AUTHORS: Veerle Baekelandt

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">SPM Addition in cells for intact cell cross-linking</div></div>

[STEPS]
?. seed cells for  in a 10 cm dish at a density of 6 x 106 cells per plate
?. treat cells for   with 10 µM freshly prepared SPM.SPM (Sigma-Aldrich, Saint Louis, MO, USA) was prepared to a final stock concentration of 200 mM in 0.1 M 3-(N-morpholino)propane sulfonic acid (MOPS)-KOH (pH 7.0)
?. wash cells with 1X PBS and scrape cells for intact cell cross linking
TITLE: No Title
AUTHORS: 

[STEPS]
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TITLE: Total Protein Extraction from Plants
AUTHORS: May Shen

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Total Protein Extraction from </span><span style = "font-weight:bold;">Plants</span></div></div>

[STEPS]
?. [New Sample Preparation]
+ finely grounded.Move into 1.5 ml eppendorf.
[plant tissue]
[2X sample buffer]
?. [New Sample Preparation]
dry bath for
65 °C
?. [New Sample Preparation]
Centrifuge: 12000 33, 1 min
?. [Old sample thawing back]
dry bath for
65 °C
?. [Old sample thawing back]
Centrifuge: 12000 33, 1 min
?. [Old sample thawing back]
Ready to load
?. [New Sample Preparation]
Ready to load or store in
-20 °C
TITLE: U Cinn - Phospholipids Assay
AUTHORS: Patrick Tso, Dana Lee

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary:  </span></div><div class = "text-block">Determinations of phospholipids in plasma/serum/lymph will be made using the Wako Phospholipids C enzymatic assay.  In this assay, phospholipids in the sample are hydrolyzed ultimately producing a blue pigment.  The amount of phospholipids in the sample is determined by measuring the absorbance of the blue color.  </div></div>

[STEPS]
?. Prepare working standards by making a serial dilution of the stock 300mg/dl standard. (See diagram below) *Stock standard included in kit.
?. Prepare Working Reagent by reconstituting one vial of Color Reagent with a portion of Buffer then transferring entire contents to Buffer bottle, rinsing Color Reagent vial several times.
?. Using a 96 well flat bottom plate, into separate wells, pipette 2μL of deionized water, standard, or sample to be assayed.
?. Add 300μL of Working Reagent to all wells.
?. Incubate plate for 5 minutes at 37°C.
?. Determine the absorbance (abs) of the standards and of each unknown at 600nm.
?. Calculate values of unknowns from the standard curve. Specimen:  Serum or Plasma.  Specimen stable for 7 days at 2-8°C or 3 months at -20°C. Assay Linearity: 1000 mg/dl Reagent Stability:  7 days at 2-8°C Stability of Final Reaction:  60 minutes
TITLE: Plant Infection with Xanthomonas Campestris Campestris 
AUTHORS: Cleo B.

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Plant infection of three week old plants with </span><span style = "font-style:italic;">Xanthomonas Campestris </span><span>Campestris. </span></div></div>

[STEPS]
?. [Day 1 ]
Make at least 40 mL of overnight culture of Xanthomonas Campestris Campestris at
28 °C
?. [Day 2]
Measure OD600 of O/N culture
?. [Day 2]
Centrifuge culture for  at
Centrifuge: 4000 33
?. [Day 2]
Remove supernatant
?. [Day 2]
Dissolve pellet in H2O until an OD600 of 1. Make sure you have at least 30 mL of diluted culture.
?. [Day 2]
Dip sterilized scissors in diluted culture and cut of the tip of a leave (about 1 cm). Make sure to cut over the central vein. Repeat this for each leaf you want to infect.
TITLE: L-3 LEECH STORAGE
AUTHORS: REDI-NET Consortium

[DESCRIPTION]
This protocol describes leech storage.

[GUIDELINES]
OBJECTIVE

To outline steps for properly storing field-collected leech samples and nucleic acid samples purified from these soil.

SUMMARY/SCOPE

The overarching aim of the REDI-NET is to develop a collaborative laboratory network between domestic and international partnering institutions to address disease surveillance needs in order to effectively detect, predict and contain potentially emergent zoonosis. This SOP provides guidance on storage of leech samples and their purified nucleic acid to preserve their integrity for downstream nucleic acid extraction and/or sequencing library preparation.


MAINTENANCE OF EQUIPMENT

Decontaminate a PCR workstation by keeping the UV light on for 15 min.
 


[STEPS]
SECTION: STORAGE PROCEDURE FOR UNTREATED SAMPLE
1. 
 
Cool 96-well microfuge tube racks on ice.
SECTION: STORAGE PROCEDURE FOR UNTREATED SAMPLE
3. Using clean forceps, transfer individual leech into the corresponding pre-labeled 2.0 mL microfuge tubes and put it onto the microfuge tube rack on ice sequentially.

SECTION: STORAGE PROCEDURE FOR UNTREATED SAMPLE
4. Once the rack is full or all leech samples have been completed, label the rack ID with a unique ID.
SECTION: STORAGE PROCEDURE FOR UNTREATED SAMPLE
5. Close the rack lid tightly, secure with clear Saran wrap and immediately transfer to -80 °C or -20 °C freezer.

 

SECTION: STORAGE PROCEDURE FOR TOTAL NUCLEIC ACID
7. 
 
In the clean PCR workstation, carefully transfer the eluted total nucleic acid to a 96-well PCR microplate, make sure to keep samples in the exact same locations corresponding to the rack where the original ticks were stored.
 

SECTION: STORAGE PROCEDURE FOR TOTAL NUCLEIC ACID
8. Cover the 96-well PCR microplate with adhesive film to prevent spill over or contamination.
SECTION: STORAGE PROCEDURE FOR TOTAL NUCLEIC ACID
9. Label the film with a unique plate ID.
SECTION: STORAGE PROCEDURE FOR TOTAL NUCLEIC ACID
10. Immediately transfer the 96-well PCR microplate to -80 °C freezer.
SECTION: STORAGE PROCEDURE FOR TOTAL NUCLEIC ACID
11. Update the freezer inventory so that samples can be tracked properly.
SECTION: STORAGE PROCEDURE FOR UNTREATED SAMPLE
2. Label 2.0 mL microfuge tubes with Sample ID.
SECTION: STORAGE PROCEDURE FOR UNTREATED SAMPLE
6. Update the freezer inventory so samples can be tracked properly.
TITLE: Qiagen DNA PowerWater DNeasy Extraction
AUTHORS: Christopher LeBoa, Sneha, Sneha

[DESCRIPTION]
These are instructions for using the Qiagen Power Water Kit for doing Extraction from drinking water samples

[BEFORE_START]
Solution PW1 must be warmed at 55°C for 5–10 minutes to dissolve precipitates.
 If Solution PW3 has precipitated, heat at 55°C for 5–10 minutes to dissolve precipitate.
 Shake to mix Solution PW4 before use.


[STEPS]
1. Insert the filter into a 5 ml PowerWater DNA Bead Tube.
2. Add 1 ml of Solution PW1 to the PowerWater DNA Bead
3. Secure the tube horizontally to a Vortex and tape it extensively
4. Vortex at maximum speed for 5 min. Centrifuge the tubes ≤4000 x g for 1 min at room temperature
5. Transfer the supernatant to a clean 2 ml Collection Tube Draw up the supernatant using a 1 ml pipette tip by placing it down into the beads.
6. Centrifuge at 13,000 x g for 1 min at room temperature.
7. Avoiding the pellet, transfer the supernatant to a clean 2 ml Collection Tube (provided).
8. Add 200 µl of Solution IRS and vortex briefly to mix. Incubate at 2–8°C for 5 min.
9. Centrifuge the tubes at 13,000 x g for 1 min.
10. Avoiding the pellet, transfer the supernatant to a clean 2 ml Collection Tube (provided).
11. Add 650 µl of Solution PW3 and vortex briefly to mix.
12. Load 650 µl of supernatant onto an MB Spin Column. Centrifuge at 13,000 x g for 1 min. Discard the flow-through. Repeat until all the supernatant has been processed.
13. Place the MB Spin Column Filter into a clean 2 ml Collection Tube.
14. Add 650 µl of Solution PW4 (shake before use). Centrifuge at 13,000 x g for 1 min.
15. Discard the flow-through and add 650 µl of ethanol and centrifuge at 13,000 x g for 1 min.
16. Discard the flow-through and centrifuge again at 13,000 x g for 2 min.
17. Place the MB Spin Column into a clean 2 ml Collection Tube
18. Add 100 µl of Solution EB to the center of the white filter membrane.
19. Centrifuge at 13,000 x g for 1 min. 23. Discard the MB Spin Column. The DNA is now ready for downstream applications.
TITLE: Colonic epithelial cell isolation for Single Cell RNA-sequencing
AUTHORS: David Fawkner-Corbett

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Protocol for isolation of single colonic epithelial cells from human endoscopic biopsies.</div><div class = "text-block"><span>Reference -  </span><a style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://doi.org/10.1038/s41586-019-0992-y</span></a></div></div>

[STEPS]
?. Biopsies transported on ice in 10ml of transport medium
?. Wash with 10ml of cold (4degree) wash medium, shake, remove media, repeat a total of 3 times
?. Transfer biopsies to 5mL warm (water bath at 37 degree) chelation medium
?. Incubate at 37C for 20mins – shake after 10mins and 20mins
?. Remove supernatant, Transfer biopsies to fresh 5ml warm chelation medium
?. Incubate at 37C for 10mins
?. Vortex 2 x 5s (or 5-10 hard shakes)
?. Allow biopsies to settle. Remove supernatant with FCS-coated pasteur, keep supernatant and replace 5ml warm chelation medium on biopsies. Return to 37C water bath.
?. Examine supernatant. If it contains crypts, spin down at 300G 4mins 4C
?. Resuspend crypt pellet in 2ml cold transport medium, place in 15ml Falcon on ice.
?. Repeat steps 5-10 and pool crypt containing fractions (3 or max 4 times)
?. Spin down pooled crypt suspension (300g, 4mins)
?. Resuspend in 3ml warm TrypLE Express with 50ug/ml DNAse at 37C
?. Incubate 45mins at 37C in incubator with agitation or rotation (e.g. MACSmix.)
?. Quench with 3ml of Transport medium supplemented with 5% FCS
?. Remove filter, wash with 15ml Transport Medium supplemented with 5% FCS
?. Remove supernatant. Wash pellet with 10ml Wash medium
?. Spin 300G 5mins.
?. Very carefully remove supernatant until est. 1ml remains. Do not disturb pellet, resuspend pellet in this.
?. Run through 30um cell strainer (prepped with FCS) and wash through with 5ml of PBS + 0.04% BSA
?. Spin 300G 4 mins, aspirate until
?. Take 10uL for count (Trypan blue, Invitrogen Countess or haemocytometer). For 10x count each sample x3  to obtain average count and viability. Ensure high viability.
?. Centrifuge (300G, 5 mins) and re-suspend using live count to concentration of 1000 cells/ul (1x10^6 / ml).
?. Process on 10x using manufacturers protocol
?. Filter with 70um cell strainer (prepped with FCS) into 50ml Falcon, wash through with 5 ml of transport medium +5% FCS
?. Spin filtrate 300G 4mins
TITLE: Priming and loading a MinION flowcell v2
AUTHORS: Josh Quick

[STEPS]
?. Thaw the following reagents at room temperature before placing on ice:  Sequencing buffer (SQB)Loading beads (LB)Flush buffer (FLB)Flush tether (FLT)
?. Add  FLT to the FLB tube and mix well by vortexing.
30 µl
?. If required place a new MinION flowcell onto the MinION by flipping open the lip and pushing one end of the flowcell under the clip and pushing down gently.
?. Rotate the inlet port cover clockwise by 90° so that the priming port is visible.
?. Take a P1000 pipette and tip and set the volume to . Place the tip in the inlet port and holding perpendicularly to the plane of the flowell remove any air from the inlet port by turning the volume dial anti-clockwise.
800 µl
Be careful not to remove so much volume that air is introduced onto the rectangular array via the outlet.
?. Load  of FLB (plus FLT) into the flow cell via the inlet port, dispense slowly and smoothly trying to avoid the introduction of any air bubbles.
800 µl
?. Wait for.
?. Gently lift the SpotON cover to open the SpotON port.
?. Load another  of FLB (plus FLT) into the flow cell via the inlet port, this will initiate a siphon at the SpotON port to allow you to load the library dilution.
200 µl
?. In a new tube prepare the library dilution for sequencing:  AB1ComponentVolume2SQB37.5 µL3LB25.5 µL4Library12 µL5Total75 µL
AB1ComponentVolume2SQB37.5 µL3LB25.5 µL4Library12 µL5Total75 µL
Mix LB immediately before use as they settle quickly. Make up with EB if less than 12 µL library is required.
?. Mix the prepared library gently by pipetting up and down just prior to loading.
?. Add the  library dilution to the flow cell via the SpotON sample port in a dropwise fashion. Ensure each drop siphons into the port before adding the next.
75 µl
?. Gently replace the SpotON sample port cover, making sure the bung enters the SpotON port, close the inlet port and close the MinION lid.
TITLE: Bulk preparation of agarose gel
AUTHORS: Callen Hyland

[DESCRIPTION]
For applications when you only need a small amount of agarose gel, it is convenient to prepare the gel in bulk ahead of time. The desired quantity of agarose can then be remelted when needed. This protocol can be modified for any agarose percentage or buffer. Fluorescent dyes for DNA visualization can be added during the remelting step.

[BEFORE_START]
This protocol is for 200 mL of 1% agarose, but it is scalable for any volume or percentage of agarose. For every 100 mL, use 1 g of agarose powder for 1%, 2 g for 2%, etc.

[STEPS]
SECTION: Bulk agarose gel prep
1. Weigh 2 g of agarose powder and add it to a beaker or Erlenmeyer flask. Use a beaker or Erlenmeyer flask that is at least 2X the volume of the agarose you are preparing.
SECTION: Bulk agarose gel prep
2. Add 200 mL of buffer to the beaker or flask and swirl to mix. For gel electrophoresis, use 1X TBE buffer. For Hydra culture experiments, use Hydra medium.

 

SECTION: Bulk agarose gel prep
3. Microwave at 80% power until the liquid starts to bubble. Using 80% power instead of full power will help prevent the molten agarose from boiling over. The length of time will vary depending on the type of microwave and the volume of liquid. For 200 mL, it takes about 2 minutes.

SECTION: Bulk agarose gel prep
4. When the liquid starts to bubble, take it out of the microwave and swirl to mix. Continue microwaving at 80% power in 30 second increments. After each 30 seconds, remove from the microwave and swirl, checking for any undissolved agarose. Continue until there are no flecks of undissolved agarose remaining.
SECTION: Bulk agarose gel prep
5. Pour the molten agarose into a flat, shallow tray or container and allow it to solidify. Use glass or heat-resistant plastic. Put the tray in the refrigerator to make it solidify faster.
SECTION: Bulk agarose gel prep
6. When the agarose has solidified and is cool to the touch, cut it into small pieces with a lab spatula and break it apart (Figure 1). Store the pieces in a tightly sealed container in the refrigerator.

 

SECTION: Remelt agarose gel
7. Weigh the needed amount of agarose gel pieces into beaker. Use a beaker that is at least 2X the volume the agarose will be when melted.
SECTION: Remelt agarose gel
8. Microwave on high power for 30 seconds then swirl to mix. Continue to microwave in increments of 30 seconds, removing each time to swirl, until the agarose is completely melted.
SECTION: Remelt agarose gel
9. If you are using a fluorescent dye for DNA visualization, add it now and swirl to mix it into the molten agarose.
SECTION: Remelt agarose gel
10. When the beaker is cool enough to touch with your hand, pour the molten agarose into the 
and allow it to completely solidify and cool to room temperature before using. The gel will appear slightly opaque when it solidifies.
TITLE: DNA extraction (BOMB)
AUTHORS: Yin-Tse Huang, Tsu-Chun Hung

[DESCRIPTION]
DNA extraction (BOMB)

[STEPS]
SECTION: Sample Collection
1. Add 200 µL of 0.5 mm beads to 2mL screw tube
  

SECTION: Sample Collection
2. Add200 µL of 1 mm beads to 2mL screw tube
 
SECTION: Sample Collection
3. Add870 µL Lysis master mix to 2mL screw tube. The final look:
 
 

SECTION: Sample Collection
4. Collect 20-50 mg of sample to 2mL screw tube

 

SECTION: Sample crush
5. Put the 2mL screw tube in mixmill for sample crush, at 3200 rpm  4 min 

 

SECTION: Centrifugation
6. Put 2mL screw tube in centrifuge for centrifugation, at this condition:10 x g, 3 min, 25 °C 
SECTION: DNA purification
7. Add 350 µL of isopropanol to the 1st well of 96 well plate

   

SECTION: DNA purification
0.20. Add 100 µL of magnetic beads (10 mg/ml) to the 1st well of 96 deep well plate 
  
 


SECTION: DNA purification
14. Add 300-500 µL of the sample (lysate) from the 1.5mL centrifuged tube to the 1st well of 96 deep well plate
  
 


SECTION: DNA purification
9. Add 400 µL of isopropanol to the 2nd well of 96 deep well plate
  

SECTION: DNA purification
10. Add 300 µL of 80% ethanol to the 3rd well of 96 deep well plate
 
SECTION: DNA purification
11. Add 300 µL of 95% ethanol to the 4th well of 96 deep well plate
    

SECTION: DNA purification
13. Add 100 µL of DEPC-treated water to the 6th well of 96 deep well plate 
 

SECTION: DNA purification
15. Put the prepared 96 deep well plate in the automated DNA extraction machine and select the BOMB protocol

SECTION: DNA purification
16. After the extraction is done, put on the 96 magnetic plate to pellet the magnetic bead residues.
  

SECTION: DNA purification
12. Add 300 µL of DDW to the 5th well of 96 deep well plate
  

SECTION: DNA purification
17. Collect 100 µL of the eluted sample (avoid getting magnetic bead) as the DNA template for downstream experiments
  

SECTION: DNA purification
8. Add 100 µL of magnetic beads (10mg/ml) to the 1st well of 96 deep well plate
TITLE: Live tracking of multiple mice in a neuromelanin-inducing PD model over several weeks
AUTHORS: Cristian González-Cabrera, Matthias Prigge

[DESCRIPTION]
 The Live Mouse Tracker (LMT) system is engineered for long-term, automated tracking and behavioral analysis of mice, integrating machine learning, computer vision, and RFID technology for simultaneous monitoring of multiple subjects. Specifically designed for the research community, LMT offers a Python-based framework for in-depth data analysis and is adept at facilitating studies involving mouse vocalizations. This cost-effective, DIY system is compatible with Windows OS and is tailored for tracking mouse behavior. Currently employed in our studies, LMT is pivotal in observing behavioral shifts across various domains such as arousal-resting, social interaction, and motor functions in a Neuromelanin-accumulating early-stage Parkinson’s Disease model utilizing the tyrosinase approach.


[STEPS]
SECTION: Animal Preparation for Recordings
1. Anesthetizing the Mouse: Anesthetize the mouse in the isofluorane induction chamber. Monitor the mouse to ensure it is properly anesthetized and does not feel pain.
Implantation Site Preparation: Gently clean and disinfect the area of the belly where the RFID probe will be implanted.
Implanting the RFID Probe: The RFID comes with a provided single-use injector. Carefully insert the injector at the predetermined site in the belly. Gently push the probe into place subcutaneously.
Finishing the Procedure: Withdraw the injector and ensure the probe is properly placed. Using your thumb and index fingers, move the RFID probe away from the injection site. Apply antiseptic to the implantation site if necessary.
Post-Procedure Care: Monitor the mouse as it recovers from anesthesia. Provide appropriate care and monitor for any signs of infection or complications.
SECTION: LMT Recordings (de Chaumont 2019)
2. 
Open Provide fresh bedding, food, and water in the LMT arena.
Make sure that the C: drive has enough space. Consider about 70GB per week of recording.
Adjust the dark/light controller according to your animal's cycle.

SECTION: LMT Recordings (de Chaumont 2019)
3. 
Open the LMT software and activate the option "calibrate" 
Adjust the arena position to fit the depicted area on the screen. Make sure that all the corners are properly aligned and the floor arena green dots are in green.
Close the LMT software.
Open the LMT software and activate the option "Live mouse Tracker" 
In the GUI choose the number of mice you will place for recordings (1-4).
Rename your experiment as needed.
Start the recordings
SECTION: End Recordings
4. Stop the recordings from the GUI.
Remove the mice and place them back in their home cage.
Transfer your data to the D: drive and delete it from C: one.
Clean up the arena and empty the water bottle.
SECTION: Data anaysis
5. Data analysis and pipelines can be found at https://github.com/fdechaumont/lmt-analysis
TITLE: Spike Detection 
AUTHORS: Bryan  Yoo, Jessica  Griffiths, Sarkis  Mazmanian

[DESCRIPTION]
Protocol for spike detection of GCaMP6F imaging data used in Yoo et al 2021

[STEPS]
1. Data sets of fluorescent values recorded at a rate of 0.206 s from GCaMP6F-expressing unstimulated neurons in the myenteric plexus of the proximal large intestine and analyzed with the MLspike software for Matlab downloaded from GitHub (https://github.com/MLspike/spikes) (Deneux et al., 2016). 
2. MLspike determines a new baseline to subtract from the raw fluorescence data to allow accurate modelling. The software uses a version of the Viterbi algorithm to obtain the most probable spike train. From the model, fluctuating baseline, model-estimated spike train, and most probable spike times were extracted. Polynomial coefficients (p2) were changed from 0.5 to 0.55 and (p3) from 0.01 to 0.03 for GCaMP6F fluorescence as recommended (Deneux et al., 2016). The minimum range for baseline (bmin) was changed from 0.7 to 0.5 as determined by observed fluorescence values.