eamag/protocols-with-steps huggingface dataset:
TITLE: Planet Microbe Semantic Web Application
AUTHORS: Kai  Blumberg

[DESCRIPTION]
Placeholder tutorial for the use of the Planet Microbe Semantic Web Application, accompanying the PhD dissertation work of Kai Blumberg.

[STEPS]
SECTION: Home Page
1. This protocol is a  temporary place holder for the protocol to use the Planet Microbe Semantic Web API. This protocol is included as part of Kai Blumberg's PhD dissertation work. 

This work is contained within this github repository: https://github.com/hurwitzlab/planet-microbe-semantic-web-analysis

Subsequent versions of this protocol will include the following sections:

1) A brief introduction and overview of the system 

2) A description of the command line options (arguments flags) for the `assemble_query.py` script. This script is a command line python script which can be used to assemble and submit a SPARQL query to the Planet Microbe RDF database.

3) An explanatory page showing how to navigate the relevant OBO ontologies (e.g., GO, NCBITaxon, ENVO) to search for ontology terms which can used in queries to discover information. 

4) A "how to" example showing how to use the system to query for all annotations of samples within a specific GO class hierarchy.

5) A "how to" example showing how to use the system to query for all annotations of samples within a specific NCBITaxon class hierarchy.

6) Documentation of several examples of R code that can be used to process and analyze the query results delivered from the system. 

Stay tuned!
-Kai
TITLE: Case - Lipid Analysis Assay by GC-mass spectrometry
AUTHORS: Henri Brunengraber

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary</span></div><div class = "text-block"><span>A known quantity of tissue / plasma is hydrolyzed and extracted after adding known amounts of internal standards: eg. heptadecanoic acid and cholesterol-d</span><span style = "vertical-align:sub;">7</span><span>. Fatty acids / cholesterol are analyzed as their trimethylsilyl derivatives using gas chromatography-electron impact ionization mass spectrometry (GCMS) (note: this protocol outlines the processing for palmitate and cholesterol; other fatty acids and sterols can be assayed using this preparation, see refs 1,2).</span></div><div class = "text-block"><span style = "font-weight:bold;">References:</span></div><div class = "text-block">
1. Triglyceride synthesis in epididymal adipose tissue: contribution of glucose and non-glucose carbon sources. Bederman IR, Foy S, Chandramouli V, Alexander JC, Previs SF. J Biol Chem. 2009, 284(10):6101-8.
</div><div class = "text-block">2. Influence of diet on the modeling of adipose tissue triglycerides during growth. Brunengraber DZ, McCabe BJ, Kasumov T, Alexander JC, Chandramouli V, Previs SF. Am J Physiol Endocrinol Metab. 2003, 285(4):E917-25.</div></div>

[STEPS]
?. Pipette 1ml KOH ethanol solution (1N KOH in 70% EtOH) for every 100mg of tissue or 100 μl of plasma use glass screw top tubes (may use less tissue/plasma)
?. Internals standards (IS): add 25 μl of 1mg/ml heptadecanoic acid (C17:0) and cholesterol-d7 for every 100μl of tissue (note: adjust added amount of internal standards by testing a representative sample)
?. Homogenize on ice with polytron homogenizer (tissue only)
?. Cover and heat for 3 hours at 85ºC
?. Pipette 50-100μl of solution into an Eppendorf tube
?. Add 50μl of 6N HCl
?. Add 300μl of Chloroform
?. Vortex and centrifuge for 2 minutes
?. Take 200μl of chloroform phase (bottom layer) and dry in GC vial at 75ºC
?. React with 60μl of TMS, cover, heat at 75ºC for 20 minutes
?. Transfer to GC insert and capGC-MS Analysis: Lipid TMS derivatives are analyzed using an Agilent 5973N-MSD equipped with an Agilent 6890 GC system, and a DB-17MS capillary column (30 m x 0.25 mm x 0.25 um). The mass spectrometer is operated in the electron impact mode (EI; 70 eV).
?. Selective ion monitoring of mass-to-charge ratios (m/z)
     a. Palmitate: M0=313-M+; IS, C:17=327
     b. Cholesterol: M0=368-M+; IS, Cholesterol-d7=375
     c. For other lipids: see references
TITLE: Preparation of Bacteria Glycerol Stocks
AUTHORS: Stephane  Fadanka, Shalo  Minette, Nadine    Mowoh

[DESCRIPTION]
This protocol is meant to provide researchers with a step by step procedure on how to prepare glycerol stocks in order to preserve and store bacteria for long term. 
Bacterial glycerol stocks are important for long-term storage of plasmids. The addition of glycerol stabilizes the frozen bacteria, preventing damage to the cell membranes and keeping the cells alive. A glycerol stock of bacteria can be stored stably at -80°C for many years and -20°C for several months.

Freezing is an efficient way of storing bacteria. Glycerol allows to reduce the harmful effect of ice crystals on bacteria which can damage cells by dehydration through the a localized increase in salt concentration leading to denaturation of proteins. Additionally, ice crystals can also puncture cellular membranes.


[STEPS]
SECTION: Preparing liquid culture of the bacteria to be stored
1. Prepare LB following this protocol depending on the desired amount of LB and subsequent number of glycerol stock tubes needed.

Using an inoculating wire loop, pick up some bacteria colonies from a culture plate and inoculate in an Erlenmeyer flask containing the LB (with the right antibiotic if applicable).

Grow the cells by incubating in an incubator at 37 °C for 180 minto obtain maximum cell growth.

SECTION: Diluting Pure Glycerol to 50% with Distilled water
2. Use a sterile measuring cylinder to measure 10 mLof distilled water and equal amount 10 mL of   into a 50ml falcon tube.

Cork the tube and shake thoroughly until the liquids are evenly mixed

SECTION: Filtering the 50% glycerol
3. Use a 20ml sterile syringe to aspirate the 50% glycerol from the falcon tube 

Plug in a 0.2µm micro filter and filter out the glycerol into a sterile falcon tube


SECTION: Aliquoting the bacterial culture into 50% glycerol and storing
4. To make 1ml of Bacteria glycerol stocks, aliquot 500µl of the filtered 50% glycerol into separate 1.5ml Eppendorf tubes (triplicates or more depending on the quantity of glycerol stocks needed.
Use sterile micropipette and tips to measure out equal volumes (500 µL) of the bacterial culture from the Erlenmeyer flask into the tubes containing the 50% glycerol (The final proportions of your glycerol stock should be 50% bacteria and 50% diluted glycerol).

Keep your thumb pressed firmly against the lid of the Eppendorf tube and  shake vigorously to make sure the 2 liquids mix completely.

Use a marker pen to label the tubes with the name of the bacterial strain and date of preparation of the glycerol stock

Store the vials in the freezer at -20°C until they are used.
SECTION: Aliquoting the bacterial culture into 50% glycerol and storing
5.
TITLE: Yale Murine TMC - Immunofluorescence Protocol
AUTHORS: Yun-Hee Youm, Yufeng Liu, Vishwa Deep Dixit

[DESCRIPTION]
Immunofluorescence staining of mouse tissues

[STEPS]
SECTION: Immunofluorescence Staining
1. Take out the slides to a slide holder and warm slides on a 37°C heater for 5-10 min.  Grouping: usually maximum two antibodies (with secondary antibody red + green) + nuclear staining for one slide.  Set negative control for each secondary antibody to detect any non-specific background.
SECTION: Immunofluorescence Staining
2. Label slides with pencil and circle section with Pap Pen (hydrophobic marker).  Move the slides into a humidified chamber.  Slides should remain in humidified chamber for all the following incubation steps.
SECTION: Immunofluorescence Staining
3. Fixation: Add one or two drops of 4% parformaldehyde in PBS on the tissue for 15-30 min.
SECTION: Immunofluorescence Staining
4. Wash with 0.5% Tween 20 in PBS (PBST) , 10 min x 2.
SECTION: Immunofluorescence Staining
5. Block with Blocking buffer: 10% host serum + 2% FBS in 0.5% PBST.  Incubate 1 hour at room temperature or 4°C overnight.
SECTION: Immunofluorescence Staining
6. Incubate with primary antibody: 1-10  μg/mL of purified antibody or 1:100 to 1:1000 dilution of anti-serum in 2% host serum + 2% FBS 0.5% PBST buffer overnight at 4°C.  Normally start with a dilution at 1:100.   For color-conjugated FC antibodies used in immunofluorescence, start at 1:50.
SECTION: Immunofluorescence Staining
7. Wash with 0.5% PBST, 10 min x 2.
SECTION: Immunofluorescence Staining
8. Incubate with secondary antibody: 1-10 μg/mL of purified antibody or 1:100-1:1000 of anti-serum in Blocking buffer (normally start at 1:200).  Incubate 1 h at room temperature (AVOID LIGHT).
SECTION: Immunofluorescence Staining
9. Wash with PBST, 10 min x 3.
SECTION: Immunofluorescence Staining
10. Incubate the slides in 0.1 μg/mL DAPI for 5 min.
SECTION: Immunofluorescence Staining
11. Wash with PBST twice before mounting.
SECTION: Immunofluorescence Staining
12. Clean the slides.
SECTION: Immunofluorescence Staining
13. Mount the slides with coverslips using anti-fade mounting medium.
SECTION: Immunofluorescence Staining
14. Seal the edges of the coverslip with nail polish and let it dry.
TITLE: No Title
AUTHORS: 

[GUIDELINES]
Ingridients stocks can be batch made and sterilize by filtering through 0.22&nbsp;&mu;m filter. Lysis buffer sets are recommended to make freshly. If you are doing ChIP routinely, you can also batch make lysis buffer sets, filter through 0.22&nbsp;&mu;m filter and store @ 4 &deg;C, protecting from light (&nbsp;sodium deoxycholate is light sensitive). Just remember, add protease inhibitors just before using.

[STEPS]
?. 
?. 
?.
TITLE: Immunocytochemistry for CASR in iPSc-derived dopaminergic neurons
AUTHORS: Peter Kilfeather

[DESCRIPTION]
CASR immunocytochemistry protocol to accompany Kilfeather, Khoo et al., 2023: Single cell spatial transcriptomic and translatomic profiling of dopaminergic neurons in health, ageing and disease



[STEPS]
SECTION: Day 1
1. Fix the iPSC derived neurons in 4% PFA.
SECTION: Day 1
2. Permeabilise cells for 10 min in a solution of 5% NDS, 1% BSA and 0.5% Triton-X 100.
SECTION: Day 1
3. Block in a solution of 5% NDS and 1% BSA for 1 h.
SECTION: Day 1
4. Incubate cells for 16 h O/N at 4°C with the following primary antibodies: CASR (Abcam Cat# ab79038, RRID:AB_2071489, 1:500), MAP2 (Abcam Cat# ab92434, RRID:AB_2138147, 1:1000),  tyrosine hydroxylase (Millipore Cat# AB1542, RRID:AB_90755, 1:500).
SECTION: Day 2
5. Wash cells twice in PBS and then incubate in secondary antibodies and DAPI for 1.5 h at RT.
SECTION: Day 2
6. Wash cells 3 times and store in fresh PBS till imaging.
TITLE: Mouse Cardiac Perfusion Fixation and Brain Collection
AUTHORS: Allen Institute for Brain Science

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes the procedures for intracardiac perfusion fixation of postnatal mice, including anesthesia, exsanguination, fixation, brain removal and post-fixation storage. </div><div class = "text-block"><span style = "font-weight:bold;">Note</span><span>: 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.</span></div></div>

[STEPS]
TITLE: 14 SDS-PAGE
AUTHORS: TJUSLS China

[STEPS]
?. Mount the gels in the vertical electrophoresis apparatus.
?. Load samples under the cathode buffer. Apply 10μL sample volumes to 0.7 × 5 mm sample wells.
10 µl
?. Set running conditions appropriate to your type of gel. We usually set 200V to run at the beginning, after 10 minutes change the voltage into 300V for another 30 minutes.
?. Protein can be visualized directly in the gel by Coomassie staining or silver staining. We usually choose Coomassie staining, then transfer the gel to the decolorizing solution and remove the dye solution.
?. Recording with gel imager.
TITLE: Rapid quantification of cellulose nanocrystals by Calcofluor White fluorescence staining
AUTHORS: Roi Peretz, Hadas Mamane,  Elizaveta Sterenzon, Yoram Gerchman

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:left">Cellulose nanocrystals (CNCs) have gained worldwide interest for their properties. CNCs are mostly produced by acid hydrolysis of cellulose-rich biomass, but its’ quantification is still complicated and tedious. Here, we describe a rapid method for the determination of CNCs concentration using Calcofluor White (CW) fluorescence dye. This method is robust, easy, selective for crystalline cellulose and suitable for routine measurement in CNCs production.</div></div><div class = "text-block"><div class = "justify" style = "text-align:left"></div></div></div>

[STEPS]
?. [Standard calibration curve]
For calibration curve, prepare different CNC solutions in DI water (up to 0.5% w/v).
?. [Standard calibration curve]
In flat-bottomed black 96 plate, place 100 µL of sample, 20 µL of 5M KOH and 80 µL of CW reagent (in this order). KOH is needed for basic conditions (pH=12-13).
?. [Standard calibration curve]
Fluorescence signal can be quantified using plate reader and report in the instrument’s Relative Fluorescence Units (RFU). Excitation is measured at 355 nm and emission at 433 nm. The plate should be continuously shaken to prevent CNC sedimentation and temperature should be kept at 30 °C.
?. [Standard calibration curve]
Fluorescence is read in kinetic mode every 10 min with maximal incubation time of 60 min until plateau is reached.
?. [CNC production from Whatman filter paper and total estimation]
Grind and dry Whatman filter paper 1.
?. [CNC production from Whatman filter paper and total estimation]
Add the ground dried filter paper sample to 64% aqueous sulfuric acid at 1:20 ratio (w/w).
?. [CNC production from Whatman filter paper and total estimation]
Incubate the mixture for 45 minutes at 45 °C.
?. [CNC production from Whatman filter paper and total estimation]
Stop the reaction by diluting the mixture 10-fold with cold water.
?. [CNC production from Whatman filter paper and total estimation]
Remove the acid from the CNCs by centrifugation (10,000 rpm) for 15 min at 10 °C.
?. [CNC production from Whatman filter paper and total estimation]
Decanting the liquid, and re-suspend the pellet in DIW for three repeating washes.
?. [CNC production from Whatman filter paper and total estimation]
Re-suspend the CNC in 30 ml DIW, and dialyze against deionized water until a constant pH is reached.
?. [CNC production from Whatman filter paper and total estimation]
Follow the CNC fluorescence procedure as mentioned above, and compare the results to the calibration curve, in order to establish CNCs concentration.
TITLE: Bacterial Transformation Protocol 
AUTHORS: Olivier George, Sierra Simpson

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Transformation of bacteria to amplify DNA for cloning, virus production, or other molecular biology techniques. </div></div>

[STEPS]
?. Transfer competent cells (DH5a) from -80 to wet ice for 5-10 min or until thawed.
?. Add 1ng to 50ng of DNA directly to cells. Incubate for 10 minutes on ice.
?. Heat shock cells for 45 seconds at 42C in a heat block. (Do not go over 45 seconds!) You can kill bacteria by keeping them at high temps for too long.
?. Transfer cells to ice and incubate for 5 minutes.
?. Add cells to sterile culture tubes with 5ml of LB + selection antibiotic. Ensure lid is not tight to ensure proper aeration of the cultures.
?. Incubate starter culture for 1h at 230rpm in a shaker at 37C.
?. Add 100ul of starter culture after incubation to warmed LB plate. Spread using roller beads.
?. Incubate plates o/n at 37C with the plate facing down to avoid desiccation of the agar.
?. In the morning, select colonies and culture in 3-5ml of Luria Broth (LB) overnight for Mini-prep in following morning.
?. Isolate the DNA from the culture.
?. Digest the plasmid with appropriate restriction enzyme and visualize with gel electrophoresis to determine if the plasmid insert is correct.
?. Send out the plasmid for sequencing before using to make virus, or functional studies.
TITLE: Simple Way to Make Homemade Cloth Masks: Fabrics Treated with NaCl and Starch Solution
AUTHORS: M Mehedi Hasan Rocky, Mohammad Amzad Hossain Bhuyan, Shamim Akhtar

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:left">This protocol describes a simple way to make a mask at home using the cotton or linen woven fabric available in wearable cloth or household napkin. Fabric is soaked in a solution containing the table salt (NaCl) and starch. Starch is used to increase the droplet absorption efficiency of fabric whereas salt is added for improving the virus deactivation system. These simple preparation and wearing techniques will help anyone to prepare a mask for safety during COVID-19 pandemic, especially for those people having masks of little avail or no avail.</div></div><div class = "text-block"><div class = "justify" style = "text-align:justify"></div><style>
							  .justify:after {
							    content: "";
							    display:inline-block;
							    width: 100%;
							  }
							</style></div></div>

[STEPS]
?. [Step by Step]
Cloth size specification: Cut out a piece of rectangular cloth sheet for a mask (width from 18 to 21 inches and length from 30 to 40 inches) from a cotton/linen woven cloth/napkin fabric. Fold over all sides ¼ inch and stitch down.
?. [Step by Step]
Clean the cloth sheet for the mask with a normal detergent or soap.
?. [Step by Step]
Salt-starching technique: For the salt-starching treatment of the cloth sheet, prepare a solution made up of ½ cup liquid starch (homemade or laundry liquid starch), 1 spoon table salt (NaCl), dissolved in 1.5 to 2-liter hot water. For good smell add 1 or 2 drops essential oil. Soak cloth sheet properly in a bowl containing the hot salt-starch solution. This prepared solution is enough for salt-starching on 4 to 5 mask cloth sheets. Press out all unabsorbed solution off the cloth sheet. Hang it outside under the sun to dry. In rainy days no salt-starching is suggested.
?. [Step by Step]
Folding technique: Fold the cloth piece to make three layers with a width of 5-7 inches depending on one’s face shape to cover nose up to lower chin. For less fiber-containing cloth, the layer can be increased by fixing the width before cutting the cloth sheet. In the folded layer sheet two sections are now defined as mid-section (‘b’) and end-section (‘a’ and ‘c’). Iron the folded fabrics.
?. [Step by Step]
Wearing technique: Place the folded mask cloth sheet around the face in a way that mid-section (‘b’) of the cloth sheet covers the nose, mouth and lower chin properly. Place two end-sections (‘a’ and ‘c’) of the cloth sheet back of the head and hold them moderate tight before making a knot at the back of your head. Other modifications such as the use of buttons may also be adopted to avoid knot style.
?. [Step by Step]
Sterilize & Reactivation: Place in boiling water for 10 minutes to sterilize and reduce the starch content. Reuse it after dry or reactivate continuing Step -2 to Step -5.
TITLE: ImmPRESS Polymer Detection Following BaseScope In Situ Hybridization
AUTHORS: madalynn.erb Erb

[DESCRIPTION]
Protocol for performing ImmPRESS immunostaining with alkaline phosphatase labeling after BaseScope In Situ Hybridization mouse brain sections.

[STEPS]
SECTION: Day 1
1. Complete BaseScope in situ hybridization using 35um mouse brain sections. Do not dry slides.
For BaseScope protocol see : (dx.doi.org/10.17504/protocols.io.5qpvo364zv4o/v1) 
SECTION: Day 1
2. Wash slides 2 times in PBS at room temp 5 minutes each wash
SECTION: Day 1
4. Incubate sections in rabbit anti TH (1:500) in 0.4% BSA, 5% horse serum and 0.1% tritonX-100 overnight at 4 degrees C
SECTION: Day 1
3. Block sections in 0.4% Bovine Serum Albumin (BSA), 10% horse serum, 0.3% triton X-100 for 1 hour at room temp
SECTION: Day 2
5. Wash slides in PBS at room temp (3 times 5 minutes each wash)
SECTION: Day 2
6. Incubate slides in secondary antibody (ImmPRESS-AP Polymer Reagent) at room temp for 2 hours.
SECTION: Day 2
7. Wash slides in PBS at room temp (3 times 5 minutes each wash)
SECTION: Day 2
8. Prepare the Alkaline Phosphatase Substrate

a. Make 2.5mL 100mM Tris-HCl, pH 8.2
b. Add 1 drop of Reagent 1
c. Add 1 drop of Reagent 2
d. Add 1 drop of Reagent 3
e. Mix well – use immediately 
SECTION: Day 2
9. Incubate sections in alkaline phosphatase substrate for 5 minutes
SECTION: Day 2
10. Wash slides in PBS (2 times 5 min each wash)
SECTION: Day 2
11. Dry slides at 60°C for ≥15min until they are completely dry
SECTION: Day 2
12. Coverslip slides using VectaMount 
SECTION: Day 2
13. Store slides at room temp
TITLE: Competitive enzyme-linked immunosorbent assay for investigating SpL binding to mammalian and avian immunoglobulins
AUTHORS: Angel  Justiz-Vaillant

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This ELISA was based on the theory that antibodies present in different samples would compete with human IgG for binding to SpL, resulting in inhibition of human IgG-SpL interactions [1].</div><div class = "text-block">Reference:</div><div class = "text-block"><span>1.  Justiz-Vaillant AA, Akpaka PE, McFarlane-Anderson N, Smikle MF. Comparison of techniques of detecting immunoglobulin-binding protein reactivity to immunoglobulin produced by different avian and mammalian species.</span><span style = "font-style:italic;">West Indian Med J</span><span>. 2013;62(1):12-20.</span></div></div>

[STEPS]
?. This ELISA is based on the theory that antibodies present in different samples would compete with human IgG for binding to SpL, resulting in inhibition of human IgG-SpL interactions.
?. The samples tested are commercially prepared pooled sera from skunk, coyote, raccoon, duck, and also commercially prepared purified immunoglobulins from cat and chicken (SigmaAldrich Co, St Louis, Missouri).
?. The microplate is coated with 50 µl of commercial human IgG (1 µg/well overnight at 4°C).
?. Serial doubling dilutions (1:4 to 1:1024) of 30 µl of each sample are made in a separate microplate to which 30 µl of the conjugate SpL-HRP diluted 1:1000 in non-fat milk is added.
?. The microplate is incubated for one hour at RT and then 50 µl of each sample is transferred to the human IgG coated microplate and incubated for one hour.
?. The microplate is then washed four times with PBS-Tween 20 buffer (SigmaAldrich Co, St Louis, Missouri), and 50 µl of the substrate OPD (3 mg/ml) is added to each well and incubated at RT for 15 minutes.
?. The reaction is stopped with 3M H2SO4 and the microplate is visually assessed and read at 492 nm.
?. The percentage of the binding inhibition (I%) of the SpL-human IgG interactions by different samples was calculated.