eamag/protocols-with-steps huggingface dataset:
TITLE: Manual Tracing Study: Tibetan Knot 
AUTHORS: Jamin Pelkey, Sari Park, Zahra Vahedi, Stephanie Walsh Matthews

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Lab Study Script and Protocols for manual tracing study using Tibetan knot pattern (Prime) and asymmetrical pattern (Anti-prime) and Control (non-tracing).</div></div>

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol was designed and developed at this laboratory.</p>
<p>The assay targets the capsid peptide coding region of DENV 1-4 and is desigend as a qualitative screening test for human cases of DENV infection.</p>

[BEFORE_START]
<div>
<ul>
<li>If using a different brand or model of real-time thermocycler, check the concentration of ROX is adequate.</li>
<li>Method assumes the user is familar with the thermocycler and software used to run the protocol.</li>
</ul>
</div>

[STEPS]
?. 
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TITLE: Tissue Fixation | HubMAP | JHU-TMC
AUTHORS: Kyu Sang Han, Pei-Hsun Wu, Joel Sunshine, Ashley Kiemen, Miklhail James, Sashank Reddy, Denis Wirtz

[DESCRIPTION]
 First and most important - the original tissue sample must be of good quality. Factors such as warm 
ischemic time, the time delay between tissue excision and fixation, etc. are important. Ideally, tissues 
should be acquired as close to still being viable as possible, and put into fixative as soon as possible
following excision. Delays lead to cell death, autolysis, and loss of tissue and cell integrity with concomitant 
losses of immunostaining (e.g., due to proteolysis of the antigen). If acquiring animal tissues, consider 
performing perfusion fixation before organ/tissue removal if it is an option

[STEPS]
SECTION: Tissue Fixation
1. Tissue is harvested using our protocol (Tissue Harvesting Protocol)
SECTION: Tissue Fixation
4. The rate of penetration of formaldehyde depends on the size of the biopsy. Trimming is often required to facilitate fixation. Tissues placed in the tissue cassettes should be no thicker than 3-4mm because the interior of a specimen may not become fully fixed, or significant autolysis can occur.

Reference - tmalab.jhmi.edu/histology.html#proc
SECTION: Tissue Trimming
5. Trimming also helps remove unwanted parts of the biopsy such as excessive fat and traumatized
edges.
SECTION: Tissue Trimming
6. Label tissue cassette with sample ID with pencil
SECTION: Tissue Trimming
7. Place the trimmed tissue in cassette
SECTION: Tissue Trimming
8. Place cassettes back into the histology container
SECTION: Tissue fixation (continue)
9. Refill 10% NBF into the container and leave for 12~48 hours at room temperature.

Use plenty of fixative. The general rule is to use at least 15 volume equivalents of formalin per volume 
of tissue. A higher formalin-to-tissue ratio certainly won't hurt, and just requires a larger container. 
Formalin is relatively cheap, so don’t skimp on this step.

Recommended reading - Active monitoring of formaldehyde diffusion
into histological tissues with digital acoustic interferometry - PMC (nih.gov)
SECTION: Tissue fixation (continue)
11. Pour the formalin into an “excess formalin” waste container in the hood.
SECTION: Tissue fixation (continue)
12. Rince the cassettes in the histology container with 1x PBS 
SECTION: Tissue fixation (continue)
13. Drain and refill the PBS. Leave the cassette submerged in PBS and store at 4 degree C
until processing.
SECTION: Tissue fixation (continue)
14. Properly fixed tissue can be stored in PBS up to a week.
SECTION: Tissue Fixation
2. We submerge the biopsy in the histology container prefilled with 10% NBF for fixation
SECTION: Tissue Fixation
3. Minimizing the warm ischemic time is critical. The time delay between tissue excision and fixation is detrimental to further analysis.

Note on the effect of delay to formalin fixation - Delay to
formalin fixation effect on breast biomarkers - PubMed (nih.gov)

SECTION: Tissue fixation (continue)
10. Gentle agitation of the tissue in the formalin during fixation maximize diffusion and reduce the poor local fixation.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
Extraction of tissue metaboilites for untargeted LC-MS on LC-QTOF.

[BEFORE_START]
The tissue, disposable pestle and 1.5 ml-centrifuge tube in liquid nitrogen were chilled in liquid nitrogen.

[STEPS]
SECTION: Metabolite extraction
?. 
SECTION: Untargeted metabolomics by LC/MS
?.
TITLE: Desalting of Peptides to Prepare for Mass Spectrometry Analysis
AUTHORS: jconsi, Ikram Isa, Alexandra Naba

[DESCRIPTION]
Prior to proteomic analysis, peptide samples are desalted and eluted with freshly prepared 50% acetonitrile, 0.1% trifluoroacetic acid, followed by concentration in a vacuum concentrator. Peptides are then resuspended in freshly prepared 5% acetonitrile, 0.1% formic acid. 



 


[STEPS]
SECTION: Column Preparation
1. Column Preparation
SECTION: Column Preparation
1.1. Take a Pierce peptide desalting spin column and remove the white tip (do not remove the screw cap of the tube). Place in a 2mL tube and spin column at   for 1 min. 

SECTION: Column Preparation
1.2. Add 300 µLof acetonitrile. Spin at  for 1 minand discard flow-through.

SECTION: Column Preparation
1.3. Repeat this step once


 

SECTION: Sample Loading
2. Sample Loading 
SECTION: Sample Loading
2.1. Place the spin column in a new low-retention 2 mL tube labeled "flowthrough".

SECTION: Sample Loading
2.2. Load 300 µL of peptide sample into the tube and spin at   for 1 min. 
 


SECTION: Sample Loading
2.3. Based on the total sample volume, if:

2.3a) More than 300 µL of sample -> place into a new 2 mL tube and load the remaining volume
2.3b)Less than 300 µL -> reload the flow-through.



SECTION: Wash
3. Wash Sample
SECTION: Wash
3.1. Place the spin column in a new low-retention 2mL-tube and load300 µLof 0.1% TFA in HPLC-grade H2O. Centrifuge at3000 x g for  1 min.  Discard wash flow-through.

SECTION: Wash
3.2. Repeat step 3.1 2 more times.
 

SECTION: Sample Elution
4. Elute Samples
SECTION: Sample Elution
4.1. Place the spin column in a new 2mL low-retention tube labeled with the sample name.
SECTION: Sample Elution
4.2. Load 300 µLof 0.1% TFA, 50% acetonitrile in HPLC-grade H2O . Spin at  for 1 min. 


 


SECTION: Sample Elution
4.3. Transfer the spin column to another 2mL-low retention tube and repeat the step. 
SECTION: Sample Elution
4.4. Pool the two elution samples from 4.2 and 4.3. These are the desalted peptides.
SECTION: Sample Elution
4.5. Store at-20 °C.

SECTION: Lyophilization and Reconstitution
5. Lyophilization
SECTION: Lyophilization and Reconstitution
5.1. To remove reagents incompatible with mass spectrometry place tubes in SpeedVac™ until completely dry.
SECTION: Lyophilization and Reconstitution
5.2. Depending on the size of the peptide pellet, resuspend samples with20 -75 µLof 0.1% formic acid, 5% acetonitrile in HPLC-grade H2O.

SECTION: Lyophilization and Reconstitution
5.3. Vortex until completely resuspended.
SECTION: Sample Loading
2.4. Spin the sample at   for 1 min. Store “FT” at-80 °C for troubleshooting purpose. 
SECTION: Lyophilization and Reconstitution
5.4. Peptide concentration can be measures using a spectrophotometer or using peptide concentration measurement kits such as:   )
TITLE: Heat-inactivation of Fetal Bovine Serum (FBS)
AUTHORS: Andreas Sagen

[DESCRIPTION]
The objective of heat inactivation is to destroy complement activity in the serum without affecting the growth-promoting characteristics of the product. Removal of complement activity from serum, such as fetal bovine serum, is not required for most cell cultures but may be necessary for cultures that are sensitive to the complement activity. Since heat inactivation of the serum may, to some extent, decrease the growth performance properties of the serum, this procedure should only be performed if required for optimal cell growth. Researchers should evaluate the applicability of heat inactivation regarding their own application.

[BEFORE_START]
Clean and sanitize everything

[GUIDELINES]
Do not use heat inactivated FBS unless necessary.

[STEPS]
SECTION: FBS heat-inactivation procedure
1. Slow thaw FBS at 4 °C overnight

 

SECTION: FBS heat-inactivation procedure
2. Aliquot FBS into 50 mL units in a sterile environment

 

SECTION: FBS heat-inactivation procedure
3. Prepare a volumetrically equal blank container with distilled water

 

SECTION: FBS heat-inactivation procedure
4. Let FBS equilibrate with the distilled water container for 30 min at Room temperature
SECTION: FBS heat-inactivation procedure
5. Clean the circulating water bath. Fill with an appropriate amount distilled water. Set temperature at 56 °C

SECTION: FBS heat-inactivation procedure
6. Set FBS aliquots and blank into preheated water bath at 56 °C for 30 min. Use a thermometer to track temperature in distilled water blank, and start timer when temperature reach 56 °C. Agitate aliquots every 5 min to ensure homogenous heating
SECTION: FBS heat-inactivation procedure
7. Remove aliquots from heat bath, and place FBS on ice or 4 °C for 30 min
SECTION: FBS heat-inactivation procedure
8. Store aliquots at -20 °C until use
TITLE: Intestinal Lamina Propria and Spleen Immune Cell Isolation  
AUTHORS: rabdelha  

[DESCRIPTION]
This protocol details isolation of immune cells from intestinal lamina propria/spleen and flow cytometry.

[STEPS]
SECTION: Isolation of Immune Cells from Intestinal Lamina Propria/Spleen
1. Dissect the small and large intestines for isolation of intestinal lamina propria cells,  place the small and large intestines immediately on ice  cold PBS. 

SECTION: Isolation of Immune Cells from Intestinal Lamina Propria/Spleen
2. Open the intestines longitudinally after removing the mesenteric fat and Peyer’s patches (small intestine), wash out the luminal contents with cold PBS. 
SECTION: Isolation of Immune Cells from Intestinal Lamina Propria/Spleen
3. Wash the tissue pieces for   10 min in 1 millimolar (mM) dithiothreitol (DTT)/PBS at Room temperature on a rocker to remove mucus, followed by a wash for 25 min in 10 millimolar (mM) EDTA/30 millimolar (mM) HEPES/PBS at 37 °C on a platform shaker (180 rpm) to remove epithelium. 

SECTION: Isolation of Immune Cells from Intestinal Lamina Propria/Spleen
4. After a 2 min wash in complete RPMI, digest the tissue in a 6-well plate for 90 min in complete RPMI with 150 U/ml (small intestine) or 300 U/ml (large intestine) collagenase VIII (Sigma) and 150 µg/µL DNase (Sigma) in a cell culture incubator (5% CO2).

SECTION: Isolation of Immune Cells from Intestinal Lamina Propria/Spleen
5. Pass the tissue digests through a    cell strainer and separate them by centrifugation (1200 x g for 20 min) using a 40/80% percoll gradient. 

SECTION: Isolation of Immune Cells from Intestinal Lamina Propria/Spleen
6. Collect the immune cells at the 40/80% interface.
SECTION: Isolation of Immune Cells from Intestinal Lamina Propria/Spleen
7. For the spleen, pass the tissue through a   cell strainer and incubate in red cell lysis buffer (Sigma) for 8 min at Room temperature. 

SECTION: Isolation of Immune Cells from Intestinal Lamina Propria/Spleen
8. Wash both spleen and intestine immune cells with 0.5% BSA/PBS before staining and fixation (eBioscience Foxp3 / Transcription Factor Staining Buffer Set).
SECTION: Flow Cytometry
9. Use , CD16/32 antibody (eBioscience) for flow cytometry staining to block the non-specific binding to Fc receptors before surface staining.
SECTION: Flow Cytometry
10. Isolate the immune cells from intestinal lamina propria and stain with antibodies against the following markers:  


 

SECTION: Flow Cytometry
11. For some panels, a lineage marker mix (Lin) contained TCRβ, B220, Ly6G and Siglec-F (PE-Cy7). 
SECTION: Flow Cytometry
12. Discriminate the live and dead cells by Live/Dead Fixable Aqua Dead Cell Stain Kit (Invitrogen).
TITLE: Halo-LC3B processing assay to assess autophagy
AUTHORS: Xuefeng Ren

[DESCRIPTION]
This protocol details Halo-LC3B processing assay to assess autophagy.

[GUIDELINES]
Reference: DOI: 10.7554/eLife.78923

[STEPS]
SECTION: Halo-LC3B processing assay to assess autophagy
1. Generating HeLa cells expressing HaloTag-LC3B using pMRX-IP-HaloTag7-LC3 from Mizushima lab (Addgene #184899; DOI: 10.7554/eLife.78923).
SECTION: Halo-LC3B processing assay to assess autophagy
2. Seed HeLa cells at 100-150K cells/well in 12-well plate one day before.
SECTION: Halo-LC3B processing assay to assess autophagy
3. Next day, incubate cells with complete DMEM medium and 50 nanomolar (nM) JF646 HaloTag ligand (Promega) for 60 min, and then wash twice with 1xPBS. The non-starved samples can be harvested immediately by trypsinization (step 5).
SECTION: Halo-LC3B processing assay to assess autophagy
4. To induce autophagy by starvation, treat cells with EBSS buffer (Gibco) for desired period.
SECTION: Halo-LC3B processing assay to assess autophagy
5. After the treatment, harvest the cells by trypsinization.
SECTION: Halo-LC3B processing assay to assess autophagy
5.1. Wash the wells with 1x PBS.
SECTION: Halo-LC3B processing assay to assess autophagy
5.2. Incubate the wells with 0.5 mL trypsin at 37 °C for 5 min.

SECTION: Halo-LC3B processing assay to assess autophagy
5.3. Add 0.5 mL complete medium into each well.

SECTION: Halo-LC3B processing assay to assess autophagy
5.4. Transfer cells into pre-chilled Eppendorf tube, spin 2000 x g, 5 min.

SECTION: Halo-LC3B processing assay to assess autophagy
5.5. Aspirate off the liquid.
SECTION: Halo-LC3B processing assay to assess autophagy
6. Resuspend the cell pellets in 30 µL of lysis buffer and incubate on ice for 30 min.

SECTION: Halo-LC3B processing assay to assess autophagy
7. Centrifuge the cell lysate at 21000 x g, 10 min. Transfer the cleared lysate into another tube, and measure protein concentration nanodrop spectrophotometer (Thermo Fisher).

SECTION: Halo-LC3B processing assay to assess autophagy
8. For each sample, load 20 µg clarified lysates onto NuPAGE 4-12% Bis-Tris Gel (Thermo Fisher).
SECTION: Halo-LC3B processing assay to assess autophagy
9. For in-gel fluorescence imaging, the gel was immediately visualized with ChemiDoc MP imaging system (Bio-Rad) after SDS PAGE. Band intensities are acquired by exciting samples at 546 nm (mCherry signal) and 647 nm (JF646 HaloTag ligand signal).
TITLE: Environmental DNA (eDNA) Sample Shipping Protocol
AUTHORS: Meghan M. Shea, Alexandria B Boehm

[DESCRIPTION]
This is a protocol for mailing PCR amplicons generated from eDNA samples, specifically via the following earlier sampling & filtration, extraction, and PCR protocols, e.g.:
 

 

 
While this protocol was designed for mailing to the Georgia Genomics and Bioinformatics Core, the general principles may be relevant to other shipping contexts.


Acknowledgements & Attributions:

Thanks to Bradley B. Tolar for guidance on successful shipping protocols. 

[STEPS]
SECTION: Preparing Samples for Shipping
1. Clean bench area with 10% bleach, 70% ethanol, and RNase Away
 

SECTION: Preparing Samples for Shipping
2. Wipe down 20 µL multichannel pipette and several PCR tube holders with RNase away. Run in UV Crosslinker for 10 minutes on each side. 
SECTION: Preparing Samples for Shipping
3. Wipe down ice bin with 10% bleach, 70% ethanol, and RNase Away, fill with ice, and add PCR products stored in 8-strip PCR tubes
SECTION: Preparing Samples for Shipping
4. Label all 96-well plates needed for storing samples accordingly
SECTION: Preparing Samples for Shipping
5. Being careful not to open PCR tubes directly over the 96-well plates, add 15 µL of sample to each well using multichannel pipette, making sure you keep track of the orientation of how the plate was filled
SECTION: Preparing Samples for Shipping
6. Seal the plates carefully with aluminum adhesive seals
SECTION: Preparing Samples for Shipping
6.1. Press around the edges of each well to make sure they are sealed
SECTION: Preparing Samples for Shipping
7. Parafilm around the edges of each plate
SECTION: Preparing Samples for Shipping
8. Wrap each plate in Kimwipes to absorb any condensation that occurs during shipping
SECTION: Preparing Samples for Shipping
9. Cut two pieces of cardboard that fit on either side of each plate; tape the cardboard around each plate to form a sandwich (to keep it from getting punctured while shipping)
SECTION: Preparing Samples for Shipping
10. Seal each plate in a new Ziploc bag and label accordingly
SECTION: Preparing Samples for Shipping
11. Bubble wrap around each plate and place in a hard container (cardboard freezer box, pipette tip box, etc.)
SECTION: Preparing Samples for Shipping
12. Seal hard container with tape and place in its own Ziploc bag; label accordingly
SECTION: Preparing Samples for Shipping
13. Place bagged box in a Styrofoam cooler that fits in an associated cardboard box and fill with ice packs
SECTION: Preparing Samples for Shipping
14. Tape Styrofoam cooler closed and secure it in the corresponding cardboard box
 

SECTION: Preparing Samples for Shipping
15. Store cardboard box at -20°C until ready to mail
TITLE: Image Registration of MALDI IMS to Microscopy
AUTHORS: Nathan Heath Patterson, Elizabeth Neumann, Jamie Allen, Danielle Gutierrez, Jeff Spraggins

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Scope:</div><div class = "text-block">To process to register MALDI IMS images to different types of microscopy images.</div></div>

[STEPS]
?. Generate a MALDI imaging mass spectrometry (IMS) pixel map using regToolboxMSRChttps://github.com/nhpatterson/regtoolboxmsrc
?. Using FIJI ImageJ, select corresponding laser ablation marks and IMS pixels in the post-acquisition autofluorescence (post-AF) and IMS pixel map, respectively.https://fiji.sc
?. Use a "Landmark Correspondences" FIJI plugin to find an affine transformation between the two images, resampling the post-AF image to the IMS pixel map by selecting post-AF image as source image, and IMS pixel map as the template image within the plugin.
?. Save the post-AF image.
?. Using regToolboxMSRC again, align other microscopy modalities (pre-IMS acquisition autofluorescence, brightfield PAS, multiplex immunofluorescence) to the newly saved post-AF image.
?. When complete, all images will be sampled in the same coordinates as the IMS pixel map.
TITLE: Behavior: Analysis Protocol
AUTHORS: Sasha Burwell

[DESCRIPTION]
This protocol details visualisation and analysis of the collected reward learning behavior data.

[STEPS]
SECTION: Analysis Protocol
1. To visualize licks around reward delivery times (ex, Fig. 2b), load a 
 
 file into the workspace and run
 

SECTION: Analysis Protocol
2. Move all the collected data from a mouse (one 
 
 file per day of behavior) into the same subfolder (ex, called ‘Data’).

SECTION: Analysis Protocol
3. Run the Matlab code 
 

SECTION: Analysis Protocol
3.1. Can run on different days during behavior, and the code will add to the analysis performed for each newly added 
 
 file.

SECTION: Analysis Protocol
3.2. Saves two data structures:


 
 – anticipatory licking, time to first lick, and trial-based locomotor data.


 
 – locomotor data across entire behavior sessions.
SECTION: Analysis Protocol
3.3. Also calculates and prints probe and anticipatory licking information.

Exclude any mouse with 
 
 of less than 0.2 (20% of the anticipatory window spent licking on the last day of training).
Exclude any mouse with 
 
 ratio of greater than 30% (high probe responses relative to their baseline tone A anticipatory licking).
SECTION: Analysis Protocol
4. Copy the 
 
 and 
 
 variables from all mice in an experimental condition into the same folder.
SECTION: Analysis Protocol
5. Run the Matlab code 
 
 to combine and extract anticipatory licking and trial-based locomotor data across all the mice (from the 
 
 variables).
SECTION: Analysis Protocol
5.1. This saves the data structure
 
 with the resized data for each mouse.

SECTION: Analysis Protocol
5.2. Also creates a variety of plots for visualization of mean and SEM.

Figure 3 is used to create the plots seen in Fig. 2d-f. 
Figure 4 is used to create the plots seen in Ext. Fig. 3a.
SECTION: Analysis Protocol
6. Run the Matlab code 
 
 on the saved 
 
 variable to extract the AUC values seen in Fig. 2g-h and Ext. Fig. 3c, d, and f.

SECTION: Analysis Protocol
7. Run the Matlab code 
 
 to combine and extract total locomotor data across mice (from the 
 
variables), as seen in Ext. Fig. 3e.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol is for isolation of high quality, high molecular weight DNA (20 kb and larger) that is suitable for PacBio library preparation. This protocol has been tested on lyophilized conidia from multiple isolates of <em>Fusarium oxysporum, </em>including f. sp. <em>apii</em>, f. sp. <em>ciceris</em>, and f. sp. <em>lycopersici</em>.</p>

[BEFORE_START]
<p>Buffered Phenol Chloroform Isoamyl alcohol (P:C:I) ((25:24:1, saturated with 10 mM Tris, pH 8.0 and 1 mM EDTA) (Sigma P2069) [Note: use stocks that are less than 3 months old]</p>

[GUIDELINES]
<ul>
<li>When pipetting solutions with high molecular weight DNA, use low retention pipet tips with ca. 2 mm of the ends removed, i.e., wide-bore tips.</li>
<li>Use low DNA-binding microfuge tubes.</li>
</ul>
<p>Mixing is critical for purification, but avoid vigorous vortexing (which shears DNA)</p>
<p> </p>
<p>We thank Martijn Rep, Benjamin Schwessinger, and Oanh Nguyen for advice. This protocol is modified from protocols from Dr. Martijn Rep, University of Amsterdam, and Dr. Benjamin Schwessinger, Australian National University.</p>

[STEPS]
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TITLE: Cost-efficient Yeast genome Flongle library 
AUTHORS: Yutaro Hori

[DESCRIPTION]
A cost-efficient protocol for constructing a Flongle library.

[STEPS]
1. Assemble the following components to end repair and add A to the genomic sample:

 
Incubate at 20 °C  for 5 min, then 65 °C for 5 min.



2. Add 50 µL  of Ampure beads (or equivalent beads) and mix well.
Wash with 75 % EtOH twice and elute with 11 µL  of 10 millimolar (mM)  Tris-HCl (pH 8.0). 
Keep the tube on the magnetic rack and do not disturb the beads while washing with 75% EtOH.
Use 1 µL of the solution to check the concentration with Qubit. The concentration needs to be at least 30 ng/uL.
3. Assemble the following components for ligation:

 
Incubate at Room temperature for 30 min.
Add 2.3 µL of 5 Molarity (m) NaCl and incubate at Room temperature for 30 min and then cfg at max speed for 20 min.
Remove sup and add PEG wash buffer, cfg at max speed for 1 min .
Remove sup and add 11 µL of 10mM Tris-HCl (pH 8.0) and incubate at 4 °C for .
Check the concentration with Qubit.

4. Assemble the following priming solution:

 
and the library mix solution:

 
. Load them and start sequencing.