eamag/protocols-with-steps huggingface dataset:
TITLE: Creating a more isogenic strain of worms starting from parental strain
AUTHORS: Cristian Riccio

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes how to create a more isogenic strain starting from a parental strain.</div></div>

[STEPS]
?. Pick a single worm into a new fresh agar NGM plate. Let it move away from the point of deposition. Pick it again into a new fresh plate. This procedure of  first picking into a plate and then into another makes sure that no other worms or eggs are co-picked with the single worm of  interest.
?. Let the single worm populate the whole plate. This will likely take a few days.
?. When the plate is full of worms, chunk the agar into two or more plates. This strain is now called  differently, for example starting with SX, the code given to the Miska lab. Alternatively, you can repeat steps 1 and 2 to increase homozygosity.
?. Freeze one of the plates. Always freeze  vials. White plug in one of the 5 coloured caps. New strain  from single worm is called SX.... . SX is the Miska lab designation in the world. If you are not working in the Miska lab, your new strain name will start with a different lab code.
?. Once a plate is full of gravid adults, bleach it and synchronise overnight.
?. Take the arrested L1s and pour them onto new plates. Grow them until they just start laying eggs.
?. Bleach a plate of synchronised gravid adults that have just started laying eggs. This will maximise the number of L1s (who are the stage that best survives cryopreservation and with a high potential of giving birth to progeny). Synchronise overnight in 2.5 ml M9.
?. Check that you have many thrashing L1s. Add 2.5 ml freezing buffer 2x and aliquot 1 ml into each of 5 cryopreservation tubes. Transfer the vials into a polystyrene box. Tape  the polystyrene box shut. Put the polystyrene box in the -80 C freezer. The box allows the temperature decrease to be  slower. The day after, put test tube in tap water for 5 min. Pour on plate and check that worms are alive after a few hours. Put other tubes in inbox  (4 tubes) and email lab manager. New strain SX.... is ready. One tube will be put in liquid nitrogen for  long term storage. Three tubes will be put in the -80 C freezer for mid term storage.
TITLE: Thawing iPSC Plate
AUTHORS: Celeste Karch, Rita Martinez, Jacob Marsh

[STEPS]
?. Determine which clones to expand based on screening.
?. Coat 24 well plate with 250-300 ul Matrigel per well (1 well per well, from 96 well).
?. Incubate at  for .
?. Remove Styrofoam box from  and remove plate.
Check on plate after   to avoid over-thawing.
?. To each well add mTesR1 supplemented with 10 uM Rock inhibitor.a. Remove desired cells from appropriate wells and transfer to tube. Spin off freezing media. Plate cells in 24 well plate.-or-b. Remove desired cells from appropriate wells and dilute 1:5 (final volume of 500uL) in mTesR1 and plate in 24 well plate.
1 ml
?. Incubate at overnight.
?. Change mTesR1 daily.
TITLE: 06 Gel purification
AUTHORS: TJUSLS China

[STEPS]
?. Column balancing step: add 500ul buffer BL to the adsorption column CA2 (the adsorption column is put into the collection tube), centrifuge at 12,000 RPM (~13,400×g) for 1 min, dump the waste liquid in the collection tube, and put the adsorption column back into the collection tube.
500 µl
?. Remove the single target DNA strip from the agarose gel (remove the excess as much as possible) and put it into a clean centrifuge tube, and weigh it.
?. Add buffer PN to the glue block (if the gel weight is 0.1g, its volume can be considered as 100 ul, then add 100 ul PN), put in 50℃ water bath, during which gently turn up and down the centrifugal tube, to ensure that the glue block is fully dissolved. If there is any undissolved glue, continue to leave for a few minutes or add more PN until the glue is completely dissolved. Put the tube on ice.
50 °C
?. Add the solution obtained in the previous step to an adsorption column CA2 (the adsorption column was placed in the collection tube), place at room temperature for 2 min, centrifuge at 12,000 RPM (~13,400×g) for 30-60 SEC, and dump the waste liquid in the collection tube, place the adsorption column CA2 into the collection tube.
?. Add 600ul bleach buffer PW to the adsorption column CA2 (check whether anhydrous ethanol has been added before use), centrifuge 30-60 SEC at 12,000rpm (~13,400×g), dump the waste liquid from the collection tube, and put the adsorption column CA2 into the collection tube.
?. Repeat step 5.
?. Put the adsorption column CA2 back into the collection tube and centrifuge at 12,000 rpm (~13,400×g) for 2 min to remove as much bleach as possible.Place the adsorption column CA2 at room temperature for several minutes to dry thoroughly to prevent the residual rinse fluid from affecting the next experiment.
?. Put the adsorption column CA2 into a clean centrifugal tube, and drop an appropriate amount of ddH2O(about 30ul) onto the middle position of the adsorption film, and leave it at room temperature for 2 min. Final DNA solution was collected by centrifugation at 12,000 RPM (~13,400×g) for 2 min.
30 µl
TITLE: Parse Evercode WT v2 -- University of Minnesota TMCs
AUTHORS: Laura J Niedernhofer, David A Bernlohr

[DESCRIPTION]
Combinatorial barcoding is an instrument-free method used to generate single-cell and single-nucleus RNA-sequencing libraries. Outlined here are the methods used to isolate single-nuclei from frozen tissue; to fix nuclei following isolation; to ligate nuclei-specific barcodes to transcript cDNA by iteratively splitting and pooling nuclei suspensions between multiple 96-well plates via the Evercode WT system; and to prepare barcoded cDNA for sequencing by synthesis (SBS). The following protocol has been adapted from protocols developed by 10x Genomics, Parse Biosciences, and Illumina to be used at the University of Minnesota TMCs in collaboration with the University of Minnesota Genomics Center. These protocols are owned by their respective companies and are subject to periodic revision.


[STEPS]
SECTION: Single Nuclei Dissociation
1.   

SECTION: Nuclei Fixation Protocol
2.   

SECTION: Evercode WT Protocol
3.   
 


 


SECTION: FASTQ Generation
4. BCL data from Illumina sequencer is demultiplexed and converted into FASTQ format using bcl2fastq version 2.20.0. 

Multiplexed FASTQ files were split by sample using a script provided by Parse Biosciences (parse_fastq_sep_groups.py, v0.4).
TITLE: Staining of CD31 and CD13 in PDGFR-B/Td-tomato brain sections
AUTHORS: Daniel Manrique-Castano

[DESCRIPTION]
This protocol is suitable for the staining of CD31 and CD13 in PDGFR-B/Td-Tomato fixed mouse brain sections.

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

Note that this protocol uses free-floating sections to enhance CD31 staining. 

This protocol uses sequential staining to avoid cross-reactivity between CD31 and CD13.

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

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


[STEPS]
SECTION: Tissue preparation and blocking
1. Extract the sections from the anti-freeze media and rinse them in PBS using a 24-well plate.

SECTION: Tissue preparation and blocking
2. Aspirate the PBS and incubate the sections in the Blocking solution for 60 min at Room temperature .



SECTION: Antibody incubation
3. When blocking is finished, aspirate the buffer (no washing is required) and incubate CD31 (BD Bioscience, 550274, 1:200) in antibody buffer for 180 min at 4 °C. 

 

SECTION: Antibody incubation
4. When primary antibody incubation is finished, aspirate the media and wash the sections 5 min with 0.05 % (v/v)    in PBS.
SECTION: Antibody incubation
5. Incubate Donkey α Goat 488 (Invitrogen, A-11055, 1:500) secondary antibody in 0.1 % (v/v)      for 60 min at Room temperature 
SECTION: Antibody incubation
8. When blocking is finished, aspirate the buffer (no washing is required) and incubate CD13 (R&D, AF2335, 1:300) in antibody buffer for 180 min at 4 Room temperature. 
 


SECTION: Wash and mounting
12. Clean the remaining buffer on the slides using absorbent tissue and mount the sections with a drop of  .

SECTION: Antibody incubation
6. When secondary antibody incubation is finished, aspirate the media and wash the sections 5 min with 0.05 % (v/v)    in PBS.
SECTION: Tissue blocking
7. Perform a second blocking step using 5% Donkey serum in 0.1 % (v/v)    in PBS. 
SECTION: Wash and mounting
11. When secondary antibody incubation is finished, wash the sections 5 min with 0.05 % (v/v)    in PBS. Follow this with 5 min washes with PBS to remove all detergent traces.  
SECTION: Antibody incubation
9. When primary antibody incubation is finished, aspirate the media and wash the sections 5 min with 0.05 % (v/v)    in PBS.
SECTION: Antibody incubation
10. Incubate Donkey α Rat 647 (Jackson, 712-605-153, 1:300) secondary antibody in 0.1 % (v/v)      for 60 min at Room temperature. Add DAPI  (Invitrogen, D3571, 1:5000)
TITLE: 10% Tween 20
AUTHORS: Allen Institute for Brain Science

[DESCRIPTION]
This protocol is used to prepare 10% Tween 20. Tween 20, when added to aqueous solutions acts as a surfactant, decreasing the surface tension of the solution. It also punches holes in membranes, allowing easier penetration of macromolecules into cells and organelles, and reduces non-specific binding of antisera in immunohistochemistry.

Note: Research reported in this publication was supported by the National Institute Of Mental Health of the National Institutes of Health under Award Number U19MH114830. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

[STEPS]
TITLE: MojoSort™ Pan DC Isolation Kit Column Protocol
AUTHORS: Ken Lau

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">MojoSort™ Pan DC Isolation Kit Column Protocol</span><span></span></div></div>

[STEPS]
?. [Protocol]
Prepare cells from your tissue of interest or blood without lysing erythrocytes.
?. [Protocol]
In the final wash of your sample preparation, resuspend the cells in MojoSort™ Buffer by adding up to 4 mL in a 5 mL (12 x 75 mm) polypropylene tube.Note: Keep MojoSort™ Buffer on ice throughout the procedure.
?. [Protocol]
Filter the cells with a 70 µm cell strainer, centrifuge at 300 x g for 5 minutes, and resuspend in a small volume of MojoSort™ Buffer. Count and adjust the cell concentration to 1 x 108 cells/mL.
?. [Protocol]
Aliquot 100 µL of cell suspension (107 cells) into a new tube. Add 10 µL Human TruStain FcX™. Mix well and incubate at room temperature for 10 minutes. Scale up the volume accordingly if separating more cells. For example, if the volume of Human TruStain FcX™ for 1x107cells is 10 µL, add 100 µL for 1 x 108 cells. When working with less than 107 cells, use indicated volumes for 107 cells.
?. [Protocol]
To make the pre-diluted biotin, mix 10 µL of the Biotin-Antibody Cocktail with 30 µL of 1X MojoSort™ Buffer. Add 10 µL of this pre-diluted Biotin-Antibody Cocktail. Mix well and incubate on ice for 15 minutes. Scale up the volume if separating more cells. For example, add 100 µL of pre-diluted Antibody Cocktail for separating 1 x 108 cells in 1 mL of MojoSort™ Buffer. When working with less than 107 cells, use indicated volumes for 107cells.
?. [Protocol]
Vortex the Streptavidin Nanobeads (to resuspend) at max speed, 5 touches. Prepare the nanobead dilution by mixing 20 µL of Streptavidin Nanobeads with 80 µL of 1X MojoSort™ Buffer. Add 20 µL of pre-diluted Streptavidin Nanobeads. Mix well and incubate on ice for 15 minutes. Scale up the volume accordingly if separating more cells. For example, add 200 µL of pre-diluted Nanobeads for separating 1 x 108 cells in 1 mL of MojoSort™ Buffer. When working with less than 107 cells, use indicated volumes for 107 cells.
?. [Protocol]
Wash the cells by adding MojoSort™ Buffer up to 4 mL. Centrifuge the cells at 300 x g for 5 minutes.
?. [Protocol]
Discard the supernatant.
?. [Protocol]
Resuspend cells in the appropriate amount of MojoSort™ Buffer and proceed to separation. At least 500 µL is needed for column separation. Note: There are several types of commercially available columns, depending on your application. Choose the one that fits best your experiment:
?. [Data]
Mouse CD4 T Cell Isolation Kit95.7%85%
Mouse CD4 T Cell Isolation Kit95.7%85%
?. [Data]
TITLE: DNA extraction (BOMB)
AUTHORS: Tsu-Chun  Hung, Yin-Tse    Huang

[DESCRIPTION]
DNA extraction (BOMB)

[STEPS]
SECTION: Sample Collection
1. Add 200 µL of 1mm beads to 1.5ml enppendorf tube

SECTION: Sample Collection
2. Add200 µL of 0.5mm beads to 1.5ml enppendorf tube

SECTION: Sample Collection
3. Add 225 µL of TE buffer to 1.5ml enppendorf tube
 

SECTION: Sample Collection
4. Add 375 µL of lysis buffer to 1.5ml enppendorf tube
 


SECTION: Sample Collection
6. Collect 10-20 mg of sample to 1.5ml enppendorf tube
SECTION: Sample crush
7. Put 1.5ml eppendorf tube in mixmill for sample crush, at this condition: 30 rpm/s, for 4mins 4 min 
SECTION: Centrifugation
8. Put 1.5ml eppendorf tube in centrifuge for centrifugation, at this condition:17.0 x g, 3 min, 25 °C 
SECTION: DNA purification
9. Add 350 µL of isopropanol to the 1st well of 96 well plate

   

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

 


SECTION: DNA purification
9.2. Add 200 µL of sample (lysate) from 15ml centrifuge microtube to the 1st well of 96 deep well plate 
 

SECTION: DNA purification
10.      Add 400 µL of isopropanol to the 2nd well of 96 deep well plate 
SECTION: DNA purification
11. Add 300 µL of 80% enthanol to the  3rd well of 96 deep well plate 
SECTION: DNA purification
12. Add 300 µL of 80% enthanol to the 4th well of 96 deep well plate 
SECTION: DNA purification
13. Add 100 µL of DD water to the 5th well of 96 deep well plate 
SECTION: DNA purification
14. Put in the automated DNA extraction machine

SECTION: Sample Collection
5. Add 267 µL of 10M ammonium acetate to 1.5ml enppendorf tube
 

SECTION: DNA purification
15. After the extraction is done, collect 100 µL of the eluted sample as the DNA template for downstream experiments
TITLE: Data File 
AUTHORS: Yolonda Freeman-Hildreth

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Data file; </div><div class = "text-block">Total sample population</div><div class = "text-block">Facebook/Social connection group</div><div class = "text-block">Amazon Mechanical Turk group</div><div class = "text-block">Qualtrics group</div></div>

[STEPS]
?.
TITLE: Crystallisation of SARS-CoV-2 N Protein
AUTHORS: Peter Marples, Lizbé Koekemoer, Daren Fearon

[DESCRIPTION]
The crystallization protocol and buffer conditions used to obtain reproducible SARS C0V-2 Nucelocapsid  crystals suitable for XChem fragment screening.

[STEPS]
SECTION: Crystallisation experiment
3. Protein and buffer requirements:
57.6 µL20 mg/mL  
2.88 mL 
5.76 µL   seeds, dilution 1:100

SECTION: Crystallisation experiment
5. Dispense 30 µL into SwissCI 3 lens plate reservoir wells using a 100 µl multi-channel pipette.
Dispense 200 nL20 mg/mL    to each lens using the SPT mosquito.
Dispense  180 nL to each lens using the SPT mosquito.
Dispense  20 nL to each lens using the SPT mosquito.

Drop ratio: 5:9:1 ratio (100 nL   : 180 nL reservoir solution: 20 nL seeds)
Final drop volume: 300 nl
SECTION: Crystallisation experiment
4. Crystallisation screen composition:
 0.1 Molarity (M) HEPES pH 7.8 
10 % isopropanol
23% w/v PEG 4000

Stock solutions used:
1 Molarity (M)  HEPES adjusted to pH 7.8  with NaOH
70 % isopropanol  
50% w/v PEG 4000

 

SECTION: Crystallisation experiment
7. Crystal form after ~12 h.
 


SECTION: Crystallisation experiment
6. Incubate at 20 °C for 1440 min in Formulatrix Rock Imager. 

Imaging Schedule: The first images are taken after 12 h and the imaging schedule follows a Fibonacci sequence of days for further collections. 
SECTION: Equipment needed
1. Formulatrix Rock Imager (or incubator of choice)
SPT mosquito
 
P100 8 multi-channel pipette
SwissCI 3 lens plate

SECTION: Crystallisation experiment
2. Prepare seed stock: 
 
1: 100 dilution    seeds
SECTION: Data collection at Synchrotron
8. Diamond Light Source
Unattended Data Collection (UDC)
Data Collection Temperature: 100K
Detector: DECTRIS EIGER2 X 9M
Beamline: I04-1
Wavelength:  0.9212 Å
Resolution (Å): 1.68
Beam Size (μm): 60 X 50
Number of images: 3600
Oscillation: 0.10°
Exposure (s): 0.0020
Transmission (%): 100
Flux (ph/s): 9.50e+11
TITLE: Anti-Neu5Gc Antibody Kit Protocol - Western Blot
AUTHORS: Sam Li

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The Anti-Neu5Gc Antibody Kit contains the essential monospecific polyclonal chicken IgY antibody, along with a negative control primary antibody to detect the presence of Neu5Gc on glycoconjugates by Western blot (WB). Samples to be evaluated are first subjected to SDS-PAGE, followed by transfer to a nitrocellulose or polyvinylidenedifluoride (PVDF) membrane. The membrane is then incubated with affinity-purified polyclonal anti-Neu5Gc to determine the presence of Neu5Gc on the protein of interest.</div><div class = "text-block">The antibody provided in this kit has been shown to identify as little as 5pmol of Neu5Gc per µg glycoprotein, which is at or below the current detection limit for conventional analysis by acid release, purification, DMB derivatization, HPLC, and electrospray mass-spectrometry. The Western blot provides additional information in that it confirms that Neu5Gc is directly linked to the glycoprotein of interest rather than to an accompanying sample component.</div></div>

[STEPS]
?. Prepare two identical SDS-PAGE gels.
?. Load and run samples in loading buffer.
?. Recommended loading of gel:Lane 1: Molecular Weight MarkerLanes 2, 8, 10: BlankLane 3-6: SamplesLane 7: Positive ControlLane 9: Negative Control
?. Confirm the presence of proteins on gel by Coomassie Staining
?. Blot proteins to membrane of choice.
?. Confirm protein transfer by Ponceau or other method of choice.
?. Block each membrane in 20ml TBS-T with 200µl diluted Neu5Gc Assay Blocking Solution, gently rocking at 4°C.
?. Incubate first blot in Primary Antibody, and one blot in Control Antibody for 2 hours at 25°C or overnight at 4°C with gentle rocking.Note: Recommended range of dilution for Western Blot is 1:1,000 to 1:10,000. The final dilution of the Primary Antibody can vary with the material being probed and the amount of Neu5Gc present. The Control Antibody should be used at the same dilution as that used for the Primary Antibody.
?. Wash blot with 50ml TBS-T for 5 min at 25°C, gently rocking 5 times.
?. Incubate blots with optimal dilution of enzyme-conjugated secondary antibody of choice at 25°C for 1 hour.
?. Wash blot with 50 ml TBS-T for 5 min at 25°C, gently rocking 5 times.
?. Develop each blot with the appropriate substrate.
TITLE: In vivo microdialysis for striatal DA release
AUTHORS: Ariadna Laguna, Miquel Vila

[DESCRIPTION]
To assess local effects of nomifensine (DAT and NET inhibitor) on striatal DA
release in microdialysis experiments

[STEPS]
SECTION: Preparation of reagents
1. Nomifensine (DAT and NET inhibitor) is dissolved in artificial cerebrospinal fluid (aCSF in mM: NaCl, 125; KCl, 2.5; CaCl2, 1.26 and MgCl2 1.18)
SECTION: Mouse surgery
3. One concentric dialysis probe (Cuprophan membrane; 6000 Da molecular weight cut-off; 1.5 mm-long) is implanted in the striatum (AP, 0.5; ML, -1.7; DV, -4.5 in mm) of isoflurane-anesthetized mice
SECTION: Preparation of reagents
2. Concentrated solutions (1 mM; pH adjusted to 6.5–7 with NaHCO3 when necessary) are stored at -80ºC and working solutions are prepared daily by dilution in aCSF. 
SECTION: Mouse surgery
4. Microdialysis experiments are performed in freely-moving mice 24h after surgery.
SECTION: Microdyalisis
5. Probes are perfused with aCSF at 1.5 μL/min
SECTION: Microdyalisis
6. Following an initial 100-min stabilization period, 5 or 7 baseline samples are collected (20 min each) before local drug application
SECTION: Dopamine levels determination
8. The concentration of DA in dialysate samples is determined by HPLC coupled to electrochemical detection (+0.7 V, Waters 2465), with 3-fmol detection limit.
SECTION: Microdyalisis
7. Nomifensine is administered by reverse dialysis at 10 and 50µM (uncorrected for membrane recovery) and then successive dialysate samples are collected
SECTION: Dopamine levels determination
9. The mobile phase containing 0.15 M NaH2PO4.H2O, 0.9 mM PICB8, 0.5 mM EDTA (pH 2.8 adjusted with orthophosphoric acid), and 10 % methanol is pumped at 1 ml/min (Waters 515 HPLC pump)
SECTION: Dopamine levels determination
10. DA is separated on a 2.6 mm particle size C18 column (7.5 x 0.46 cm, Kinetex, Phenomenex) at 28°C. 
SECTION: Data representation
11. Microdialysis data is expressed as femtomoles per fraction (uncorrected for recovery) and are shown as percentages of basal values (individual means of 5-7 pre-drug fractions).
TITLE: CODEX® Multiplexed Imaging | Modality Overview
AUTHORS: Diane  Saunders, Conrad Reihsmann, Alexander Hopkirk, Marcela Brissova, Alvin C. Powers

[DESCRIPTION]
This is an overview of all protocols currently in use by the Vanderbilt Diabetes Research Center Islet & Pancreas Analysis (IPA) Core and Powers/Brissova Research Group for performing multiplexed imaging of the human pancreas using the CO-Detection by indEXing (CODEX®) platform (now PhenoCycler™; Akoya Biosciences). It includes links to each of the individual protocols that make up the workflow for various applications and projects.

[STEPS]
1. Procure and/or prepare samples fixed with 4% paraformaldehyde (PFA).

HuBMAP | VU TMC Eye/Pancreas | Human Pancreas Processing for Multiple Applications (HuBMAP)
Mouse Pancreas Dissection and Fixation for Cryosectioning (IPA Core)

 

2. Prepare coverslips with pancreas sections.

 

ⓘ Samples mounted on coverslips can be stored at -80 °C for up to 2 months. All of our optimization of this protocol was done on   cryosections.

3. Design antibody panel using a combination of preconjugated or custom-conjugated primary antibodies.

 

ⓘ For information on HuBMAP Organ Mapping Antibody Panels (OMAPs), please visit the CCF Portal here.

4. Label tissue with primary antibodies and prepare reagents for imaging.
 

ⓘ See also: Akoya Biosciences PhenoCycler™ User Manual

6. Process raw imaging data.

As of May 2022, raw imaging data is processed using CODEX Processor v1.8.1.9 (Akoya Biosciences), which performs image stitching, z-plane selection, background subtraction, deconvolution, and shading correction.

ⓘ See also: CODEX® Processor User Instructions & Technical Notes

7. [Optional] Perform registration with other imaging modalities.

Autofluorescence (AF) Registration (HuBMAP)
 

5. Perform cyclic fluorescent imaging of tissue.