eamag/protocols-with-steps huggingface dataset:
TITLE: CXCR4 CXCR7 effects in melanoma &amp;  melanocytes
AUTHORS: Maria Elena de Bellard

[DESCRIPTION]
Chemokines are small signaling proteins released by cells in response to chemical stimuli in their environment. The chemokine stromal cell-derived factor-1 (SDF1/CXCL12) plays a role in the growth and metastasis of multiple cancers, including melanoma. SDF1/CXCL12 has two known receptors: CXCR4 and CXCR7. CXCR4 receptor signaling influences many cell responses, including the migration of neural crest cells and the amount of receptor expression upregulation in melanoma. CXCR7 receptor signaling influences melanocyte migration and constrains melanoma tumor growth in vivo, while CXCR4 receptor signaling does not. It is not known, however, if CXCR7 directly affects melanocyte and melanoma cell migration. Here, CXCR4 and CXCR7 receptor signaling were directly studied in vitro in human primary melanocytes and mouse B16-F10 melanoma cell line for migration when both receptors, individually and in combination, were genetically silenced. The silencing of CXCR7 clearly resulted in statistically significant inhibition of migration of both melanocytes and B16-F10 melanoma cells while the effects of CXCR4 silencing of migration inhibition was less clear. These results suggest that the CXCR7 receptor is more relevant than the CXCR4 receptor for the migratory capabilities of melanocytes and B16-F10 melanoma cells. These findings support down regulation or inhibition of the CXCR7 receptor through targeted therapies may be beneficial in melanoma treatment.

[STEPS]
TITLE: Confirmation of Gene Knockdown with RT-qPCR
AUTHORS: Megan Lee, Neal Bennett, Ken Nakamura

[DESCRIPTION]
This protocol confirms knockdown of individual genes from CRISPRi sgRNAs using RT-qPCR. Details below specifically validate gene knockdown of complex subunits within the electron transport chain in K562 cells.


[STEPS]
SECTION: Prepare Cell Lysis
1. Collect cell pellet, wash cells in cold PBS (For K562 cells, optimized cell number is 100.000). Place cells on ice.
SECTION: Prepare Cell Lysis
2. For each reaction, prepare 0.5 μL DNase I with 49.5 μL of Lysis solution.
SECTION: Prepare Cell Lysis
3. Add 50 μL of Lysis solution containing DNase I to cell pellet. Mix by pipetting up and down (avoid bubble). Incubate at RT for 5min.
SECTION: Prepare Cell Lysis
4. Pipette 5 μL of Stop solution into each reaction. Mix by pipetting up and down (avoid bubble). Incubate at RT for 2 min.

SECTION: Prepare Cell Lysis
5. Use sample for reverse transcription reaction or store at -20oC. Do not keep sample at RT for longer than 20 min.

SECTION: Reverse Transcription
6.  Prepare 40 μL of master mix for each reaction, place on ice
25 μL 2X RT Buffer
2.5 μL 20X RT Enzyme Mix
12.5 μL Nuclease-free Wate
SECTION: Reverse Transcription
7. Add 10 μL of cell lysis sample to each reaction. (can scale up or scale down depending on cell number, but the total volume of cell lysis sample cannot exceed 22.5 μL).

SECTION: Reverse Transcription
8. Spin down, mix gently by tapping.
SECTION: Reverse Transcription
9. Program thermal cycler for standard reverse transcription cycle (37°C for 6 minutes, followed by 95°C for 5 minutes and holding at 4°C).

SECTION: Reverse Transcription
10. Store samples at -20°C.
SECTION: Real-time PCR
11. Prepare 16 μL PCR cocktail master mix for each reaction, place on ice
10 μL TaqMan Gene Expression Master Mix (2X)
1 μL TaqMan Gene Expression Assay (20X)
5 μL Nuclease-free water

 

SECTION: Real-time PCR
12. Pipette PCR cocktail master mix to 386-well plate, add samples from RT reaction (4 μL each), avoid bubble. Put plate on ice if this process takes a long time.

SECTION: Real-time PCR
13. Cover plate with clear plastic sticker. Spin down the plate briefly.
SECTION: Real-time PCR
14. Use standard Real-time PCR program (UDG incubation of 50°C for 2 minutes, enzyme activation at 95°C for 10 minutes, and PCR cycles of 95°C for 15 seconds, 60°C for 1 minute, repeated for 40 cycles).

SECTION: Real-time PCR
15. After run is complete, calculate fold changes in expression using the 2-ΔΔCTmethod.
TITLE: Cell DIVE Platform | Accessing HuBMAP Datasets and Analysis on Globus
AUTHORS: Liz McDonough, Christine Surrette

[DESCRIPTION]
This method describes navigating to Globus from the HuBMAP data portal to access Cell DIVE data and analysis. 

[STEPS]
1. Click on the DOI link provided, or alternatively navigate to http://dx.doi.org, enter the DOI in the text box, and click Go.
2. If the DOI takes you to a Collection on the HuBMAP Data Portal, click on a HuBMAP ID of interest, located under the Dataset heading.

  

3. On the Dataset page, scroll down to the Files heading and click the provided link to Globus.

   

4. Log in to Globus using your existing organizational login e.g., university, national lab, facility, project, or eRA Commons ID.


  

5. The Cell DIVE datasets are organized under two top level directories, data_directory and extras. The data_directory contains OME-TIFF data organized by region.  The extras folder contains additional files including virtual H&Es and analysis data.

  

6. To access analysis, click through to the extras/Seg_Reg sublevel directory.  Analysis is organized by biomarker.
TITLE: Comparison of measured LDL cholesterol with calculated LDL-cholesterol using the Friedewald and Martin-Hopkins formulae in diabetic adults at Charlotte Maxeke Johannesburg Academic Hospital/NHLS Laboratory
AUTHORS: Mogomotsi Dintshi, Ngalulawa Kone, Siyabonga Khoza

[DESCRIPTION]
Background

National Cholesterol Education Programme Adult Treatment Panel III (NCEP ATP III) and the European Society of Cardiology recommends using low-density lipoprotein cholesterol (LDL-C) as a treatment target for cholesterol lowering therapy. The Friedewald formula underestimate LDL-C in non-fasted and hypertriglyceridemia patients. This study aimed to compare measured LDL-C to calculated LDL-C in diabetic patients using the Friedewald and Martin-Hopkins formulae.

Methods

The data of 1 247 adult diabetes patients were retrospectively evaluated, and included triglycerides (TG), LDL-C, total cholesterol, and high-density lipoprotein cholesterol that were measured on the Roche Cobas® c702. Passing-Bablok regression analysis was used to determine the degree of agreement between measured LDL-C and calculated LDL-C using both formulae. The Bland-Altman plots were used to assess the bias at medical decision limits based on the 2021 European Society of Cardiology (ESC) guidelines on cardiovascular disease prevention in clinical practice. 

Results

Both formulae showed a good linear relationship against measured LDL-C. However, the Martin-Hopkins formula outperformed the Friedewald formula at LDL-C treatment target <1.4mmol/L. The Friedewald formula and the Martin-Hopkins formula had 14.9% and 10.9% mean positive bias, respectively. At TG-C ≥1.7 mmol/L, the Martin-Hopkins formula had a lower mean positive bias of 4.2 % (95 % CI 3.0-5.5) compared to the Friedewald formula, which had a mean positive bias of 21.8 % (95 % CI 19.9-23), which was higher than the NCEP ATP III recommended total allowable limit of 12%.

Conclusion

The Martin-Hopkins formula performed better than the Friedewald formula at LDL-C of 1.4 mmol/L and showed the least positive bias in patients with hypertriglyceridemia.

[STEPS]
TITLE: Kompetitive Allele Specific PCR (KASP) with BioRad Software
AUTHORS: Olivia E Todd, Eric L Patterson

[DESCRIPTION]
A short guide to primer design with HEX/FAM tags and a basic KASP protocol using LGC Genomics KASP master mix and BioRad analyzation software.

[STEPS]
SECTION: Primer Design
1. Design regular primers over the mutation you wish to test.
 

SECTION: Primer Design
2. Copy and paste the FAM and HEX tags to your wild type and mutant alleles. Pay attention to which tag is which.

FAM tag: GAAGGTGACCAAGTTCATGCT
HEX tag: GAAGGTCGGAGTCAACGGATT
SECTION: Example: IAA16 GG to RR mutation Primers in Bassia scoparia
3. 
 

SECTION: Example: IAA16 GG to RR mutation Primers in Bassia scoparia
4. Thaw primers; one reverse primer, two forward primers, each specific to a SNP and corresponding fluorophore.

SECTION: Example: IAA16 GG to RR mutation Primers in Bassia scoparia
5. Make primer mix:  
      EACH forward primer    18 μl      Reverse primer  45 μl      H2O  69 μl      Total primer mix  150  
 
SECTION: Example: IAA16 GG to RR mutation Primers in Bassia scoparia
6. Add 12 µL of primer mix to the 2x KASP tube, this now the KASP master mix.

SECTION: Example: IAA16 GG to RR mutation Primers in Bassia scoparia
7. Add 4 µL of KASP master mix to each sample well of a 96 well plate.

SECTION: Example: IAA16 GG to RR mutation Primers in Bassia scoparia
8. Add 4 µL of template DNA @ 5 ng/μl-20 ng/μl.

SECTION: Example: IAA16 GG to RR mutation Primers in Bassia scoparia
9. Include 3 NTCs, as well as appropriate controls.
SECTION: Bio-Rad machine and software
10. Set up thermocycling conditions according to the protocol: 
     Step      Temperature      Length        1      94 C      15 minutes        2      94 C      20 seconds          3      61 C      1 min        4      Go to step 2       10x        5      94 C      20 seconds        6      55 C      1 min        7      30 C      10 seconds        8      Go to 5      35x   (take read each cycle)   
 
SECTION: Bio-Rad machine and software
11. Make sure that each well of the plate has both FAM and HEX fluorophores selected.
SECTION: Bio-Rad machine and software
12. Click Start Run.
SECTION: Analysis
13. On the “Allelic Discrimination” tab the relative florescence units (RFU) for FAM and HEX are displayed, these are used to make a call on the SNP(s) present in each sample.
SECTION: Analysis
14. This can be compared to the controls for identification of genotype or species. 
Example output of allelic discrimination:
TITLE: Neural activation of the GI tract
AUTHORS: Bryan  Yoo, Jessica  Griffiths, Sarkis  Mazmanian

[DESCRIPTION]
Protocol for neural activation of the GI tract used in Yoo et al 2021

[STEPS]
1. TH-Cre and ChAT-Cre mice are used for these experiments. 
2.  “Activated” mice are infected with AAV-PHP.S:hSYN1-DIO-hM3Dq-mRuby2 and “Control Mice” are infected with AAV-PHP.S:hSYN1-DIO-mRuby2. This is to control for both AAV-PHP.S-mediated expression and the effects of Compound 21 dihydrochloride (C21) (HelloBio, Princeton, NJ-HB6124). 
3. C21 was injected intraperitoneally at a dose of 3mg/kg in both activated and control mice. Mice for time course experiments were single-housed in sterile cages with autoclaved water following first C21 administration (C21 administered for 10 consecutive days). Injections of C21 we
4. Mice for time course experiments were single-housed in sterile cages with autoclaved water following first C21 administration (C21 administered for 10 consecutive days).Injections of C21 were administered at the same time every day (10AM).
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Protocol to use ACCEL-NGS® METHYL-SEQ DNA LIBRARY KIT (NGS Prep with Bisulfite-Converted DNA Capability; Cat. No. DL-ILMMS-12/48) to perform MethylC-sequencing in Arabidopsis.</p>
<p> </p>
<p>Code used to analyse raw sequencing data can be found: Please see https://github.com/dtrain16/NGS-scripts/tree/master/MethylC.</p>

[BEFORE_START]
<p>Make sure you have fresh nuclease-free water (or low EDTA TE) and 80% ethanol. You will also need working magnetic beads stocks (e.g. AMPure, SPRIselect) and a magnetic stand that holds plates or eppendorf tubes (depending on preference).</p>
<p> </p>
<p>Extract high-quaity gDNA using method of choice, our lab relies largely on the DNeasy Plant Mini Kit (Qiagen). However, yields from this are quite low despite good quality gDNA.</p>
<p> </p>
<p>For all resuspension steps, use low EDTA (0.1 mM) Tris (10mM, pH 8). Alternatively, we also use the elution buffer provided in the DNeasy Plant Mini Kit (Qiagen) - which is probably the same.</p>

[STEPS]
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TITLE: Pelvic nerve implantation, testing and processing in awake rats
AUTHORS: James Fallon, Sophie Payne, Peregrine Osborne, Janet Keast

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This collection describes the procedures required to implant a pelvic nerve array and bladder catheter into male Sprague-Dawley rats as well as cystometry and electrophysiological testing in awake animals.  </div><div class = "text-block">This collection includes protocols for:</div><div class = "text-block">STAGE 1: Implantation of a pelvic nerve array in rats</div><div class = "text-block">STAGE 2: Cystometry in awake rats</div><div class = "text-block">STAGE 3: Electrophysiological recording of electrically-evoked compound action potentials </div></div>

[STEPS]
TITLE: Phage screening - colorimetric test
AUTHORS: Kesia da Silva, Jason Andrews

[DESCRIPTION]
Phage Screening - Colorimetric Test protocol involves a series of steps designed to detect the presence of specific bacteriophages through a colorimetric assay. This comprehensive procedure aims to provide a robust method for screening      and assessing bacteriophage activity within microbial cultures, essential for various research and diagnostic applications.

[STEPS]
SECTION: Day 1
1. Prepare LB-agar plates.
SECTION: Day 1
2. Prepare LB-broth.
SECTION: Day 1
3. To recover bacteria from stock open the tube and use a sterile loop, toothpick or pipette tip to scrap off some bacteria and streak on appropriate media plates using the overnight culture and grow at37 °C. 



SECTION: Day 2
5. Next, start overnight cultures by inoculating fresh liquid media (5 mL ) with a single colony and
grown for 14-16 hours at 37 °C in a shaker incubator. 
 

SECTION: Day 3
6. Inoculate 5 mL of fresh LB (1% inoculum) and grow at 37 °C  until the cells reach a density of OD600=0.2. The amount of host culture to add is not critical, but it should be enough that would normally produce a saturated culture over the period of the enrichment (0.2 OD is enough or 0.5 McFarland Standard i.e. 1.5x 10^8 CFU/ml).
 


SECTION: Day 3
7. In a new falcon tube add 1 mL of filtered   sample to 900 µL of LB-broth and inoculate with 100 µL of a fresh host culture (0.2 OD) and mix gently. 

 

SECTION: Day 3
8. Incubate this enrichment culture at 37 °C  for at least 2 hours (or under whatever conditions the host favors). At the same time inoculate a new tube with 5 mLof fresh LB (1% inoculum) and grow at37 °C for 2 hours.

SECTION: Day 3
9. Filter the culture through a 0.22μm syringe filter. The filtrate is now ready for testing.

SECTION: Day 3
10. In a new falcon tube add 100 µL of sample (filtrate from previous step) to 1800 µLof LB-broth and inoculate with 100 µL of a fresh host culture (0.2 OD) and add 50 µL of    (resazourin) and mix gently. 

SECTION: Day 3
11. Incubate the culture at 37 °C for 3 hours. Check the culture every hour to monitor color change.


SECTION: Day 3
12. If the sample is positive for phage culture will remain blue. If the sample is negative culture will change to pink color. 

SECTION: Day 1
4. If using lyophilized strain skip step 3 and 5 and    

SECTION: Day 3
6.1. Using a pipette, aseptically add 500 µL of the recommended growth medium to the freeze-dried material and mix well.

SECTION: Day 3
6.2. Transfer the entire suspension to a test tube containing 5 mL  of the recommended media.

SECTION: Day 3
7.1.  In another falcon tube inoculate a positive control adding 10-50uL (this amount will depend of the concentration of your phage stock) of phage stock to 1900 µL  of LB-broth with 100 µL  of a fresh host culture and mix gently.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>One of the challenges to describing the human liver on a single cell level is the process of cell dissociation. Cell dissociation methods can be quite damaging to the cells and can lead to a biasing of the transcriptome. We have developed gentle surgical and perfusion techniques to isolate and transcriptionally profile viable resident cells from liver samples taken from deceased donor liver grafts prior to organ transplantation. </p>

[BEFORE_START]
<p>Prior to collagenase and neutral protease digestion, solution 3 should be warmed to 37 degree celsius.</p>
<p>Before starting any perfusions, all solutions must be oxygenated with 95%Os<sub>2 </sub>and 5% CO<span style="font-size: xx-small;">2.  </span></p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
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?.
TITLE: PBMC- 05 - In Vitro Culture of TEFF+TREG - Cytokine Production by TEFF
AUTHORS: Marco Cosentino, Elisa Storelli, Alessandra Luini, Massimiliano Legnaro, Emanuela Rasini, Marco Ferrari, Franca Marino

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">List of published work using this procedure:</div><div class = "text-block"><ul style = "list-style-type:disc;"></ul></div></div>

[STEPS]
?. Isolate TEFF and TREG with Miltenyi Kit according to the protocol PBMC- 03.
?. Count both TEFF and TREG following the appropriate protocol (CELL COUNT- 02 or CELL COUNT- 03).
?. Use sterile 96-well round bottom plates.
[Consider that these plates can contain a volume of maximum 250µL]
?. Centrifuge TEFF and TREG at 
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Centrifuge: 1200 34, 5 min
?. Resuspend TEFF and TREG in SOLUTION- 13 at a concentration of 1x106/mL.
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?. According to the experimental design, activate a desired number of wells containing TEFF cells with PHA 5µg/ml (final concentration) and IL-2 40 ng/mL (final concentration) by diluting the stock aliquots. Leave also wells of TEFF unstimulated (resting control).
?. Put TEFF-CPD labeled cells and TREG cells in the 96-well plate at  a ratio of 1:1(for example, 0.1x106 TEFF + 0.1x106 TREG) and activate the cells il the well directly (see step 7 for concentrations): include 1 control co-culture (not treated with test substance) and treated co-cultures (+test substance) according to your experimental design.
?. Include also a culture of resting and activated TEFF alone (for example 0.2x106per well), as control for the subsequent ELISA test.
?. Put the plate in a  incubator for 48 hours.
37 °C
?. At the end of cell culture, collect the supernatant in eppendorf tubes (1.5mL) and centrifuge them at in order to eliminate any residual cells.
Centrifuge: 1200 34, 5 min
?. Collect supernatants and store them at -80°C until ELISA assay is performed.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p><strong>Results of the additive and multiplicative diversity partitioning of gobiid fishes</strong></p>

[GUIDELINES]
<p>To obtain additive and multiplicative alpha and beta diversity, we used PARTITION 3; here can download manual and program: http://www.users.miamioh.edu/cristto/partition.html</p>
<p> </p>

[STEPS]
TITLE:     ELISA for quantification of IL-14 in human serum.
AUTHORS: Angel  Justiz-Vaillant

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Interleukins (IL) are a type of cytokine first thought to be expressed by leukocytes alone but have later been found to be produced by many other body cells. They play essential roles in the activation and differentiation of immune cells, as well as proliferation, maturation, migration, and adhesion. They also have pro-inflammatory and anti-inflammatory properties. The primary function of interleukins is, therefore, to modulate growth, differentiation, and activation during inflammatory and immune responses. Interleukins consist of a large group of proteins that can elicit many reactions in cells and tissues by binding to high-affinity receptors in cell surfaces. </div><div class = "text-block"><span>T cells produce IL-14, and its principal effects are stimulation of activated B cell proliferation and inhibition of immunoglobulin secretion. </span><a href="https://www.ncbi.nlm.nih.gov/books/NBK499840" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">[1]</span></a></div><div class = "text-block">Reference</div><div class = "text-block"><span>1. Justiz Vaillant AA, Qurie A. Interleukin. In:</span><span style = "font-style:italic;">StatPearls</span><span>. Treasure Island (FL): StatPearls Publishing; June 12, 2019.</span></div></div>

[STEPS]
?. An anti-human IL-14 coating antibody is adsorbed onto microwells by incubation overnight at 4°C.
?. Add 50 µl of human serum. Human IL-14 present in the serum sample binds to antibodies adsorbed to 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-human IL-14 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 IL-14 antibody. The optimal dilution of this conjugate must be investigated.
?. The plate is washed following incubation to remove the unbound Streptavidin-HRP.
?. Add 100 µl of 3',3',5',5'- tetramethylbenzidine (TMB; Sigma-Aldrich) to each well.
?. Incubate the microwells in the dark for 15 min.
?. A colored product is formed in proportion to the quantity of human IL-14 present in the sample or standard.
?. The reaction is terminated by addition of 100 µl 3M H2SO4  and absorbance is measured at 450 nm.
?. A standard curve is made from 7 human IL-14 standard dilutions and the human IL-14 sample concentration is determined.
?. For better results place the microplate on a plate shaker in every incubation.