eamag/protocols-with-steps huggingface dataset:
TITLE: Ex vivo culture of  SPMs
AUTHORS: michela.deleidi, Bianca Marchetti, Federico Bertoli, Carmela Giachino

[DESCRIPTION]
Ex vivo culture of spleen derived macrophages.

[STEPS]
SECTION: Ex vivo culture of spleen derived macrophages
1. Dissect spleens from abdominal cavity and filter it through a 40-μm nylon strainer. 
SECTION: Ex vivo culture of spleen derived macrophages
3. Then, obtain a single splenic cell suspension. Culture cells in Roswell Park Memorial Institute (RPMI) medium 1640 RPMI 1640 (BioConcept 1-41F01-I) supplemented with 10% FBS, 2 millimolar (mM) L-Glutamine and antimicrobials (Penicillin-Streptomycin Pen 10'000 IU/ml Strep 10 mg/mL and amphotericin B (250 µg/mL BioConcept). 

SECTION: Ex vivo culture of spleen derived macrophages
4. Culture mouse microglia BV2 cells from Elabscience (No.: EP-CL-0493) in parallel for each spleen culture preparation as controls. 
SECTION: Ex vivo culture of spleen derived macrophages
5. Briefly, maintain BV2 cells in Roswell Park Memorial Institute (RPMI) medium 1640 supplemented (BioConcept 1-41F01-I) with 10% FBS (FBS-02-0500), 2 millimolar (mM) L-Glutamine 5-10K50-H) and antimicrobials (Penicillin-Streptomycin Pen 10'000 IU/ml Strep 10 mg/mL and amphotericin B (250 µg/mL) (BioConcept 4-01F00-H). 
SECTION: Ex vivo culture of spleen derived macrophages
6. Spleen macrophages (SPMs) differentiate into the M1 phenotype after stimulation with LPS (100 ng/ml) ± IFN-γ (10 ng/mL). 
SECTION: Ex vivo culture of spleen derived macrophages
7. For BV2 stimulation, replace RPMI by Dulbecco’s Modified Eagle Medium (DMEM) High Glucose. (BioConcept, 1-26F03-I).
SECTION: Ex vivo culture of spleen derived macrophages
2. Use red cell lysis buffer to remove red cells.
TITLE: In situ Hybridization for Tyrosinase
AUTHORS: Ariadna Laguna, Miquel Vila

[DESCRIPTION]
In situ hybridization protocol with DIG-labelled riboprobes

[STEPS]
SECTION: Brain sectioning
1. Paraformaldehide perfused brains are cryoprotected in 30% sucrose and frozen with cold isopentane.
 
SECTION: Brain sectioning
2. Frozen brains are cryosectioned at 30 microns in a cryostat
SECTION: Brain sectioning
3. Cryosections are mounted on superfrost slides and dried over-night at room temperature before storing them at -80ºC until processing
SECTION: In situ hybridization
4. Sections are fixed for 10 min in 4% paraformaldehyde
SECTION: In situ hybridization
5. After washing in PBS, sections are permeabilized with Proteinase K for 5 min (1 μg/mL Proteinase K,
50mM Tris·HCl, and 5 mM EDTA)
SECTION: In situ hybridization
6. Refix in 4% paraformaldehyde for 10 min
SECTION: In situ hybridization
7. After rinsing in PBS, proceed with the acetylation step for 10 min with 590
mL of H 2O, 8 mL of triethanolamine, 1 mL of 37% HCl, and 1.5 mL of acetic
anhydride
SECTION: In situ hybridization
8. Incubate for 3 h at room temperature for
prehybridization (50% formamide, 5× SSC, 5× Denhardt’s solution, 250 μg/mL Baker’s yeast RNA, and 500 μg/mL salmon sperm DNA)
SECTION: In situ hybridization
9. Hybridization overnight at 72 °C in
120 μL prehybridization mix
with 2 μL of
digoxigenin-labeled sense (control probe not complementary) and antisense (test
probe complementary) probes for tyrosinase (Riboprobes are generated by PCR and labelled with
the DIG RNA labelling kit from Roche (11175033910))
SECTION: In situ hybridization
10. Wash in 72 °C heated solutions of 5× SSC and 0.2×
SSC, and then equilibrated in B1 buffer [0.1 M Tris·HCl (pH 7.5) and 0.15 M
NaCl]
SECTION: In situ hybridization
11. Preincubation for 1 h in 10% heat-in-activated FCS
in B1 buffer
SECTION: In situ hybridization
12. Incubate overnight with antidigoxigenin-AP Fab
fragments (Roche, diluted 1:5,000 in B1 with 1% heat-inactivated FCS)
SECTION: In situ hybridization
13. Wash in B1 buffer and placed the sections in a
solution of 0.1 M Tris·HCl (pH 9.5), 0.1 M NaCl, and 50 mM MgCl 2 (B2 buffer)
SECTION: In situ hybridization
14. mRNA localization is visualized after overnight incubation with 0.2 mM NBT/BCIP (Roche) and 0.24 μg/mL levamisole in 10% PVA/B2 solution protected from light
SECTION: In situ hybridization
15. The color reaction is terminated by water and mounted in Aquatex (Merck)
SECTION: In situ hybridization
16. Sections are analysed and photographed with a microscope coupled to a camera
TITLE: Determining biofilm growth amount (absorbance) 
AUTHORS: An.Huang

[DESCRIPTION]
This protocol describes a method to determine the growth amount of biofilm at the early stage of biofilm formation by measuring absorbance. Here we use our own engineered bacteria, and it requires induction of IPTG and cultured with silver ion. You may use a different kind of bacteria and different culturing medium.

[STEPS]
SECTION: IPTG induction
1. 
Escherichia coli grown overnight was diluted by LB to OD600=0.4-0.6.
SECTION: IPTG induction
2. IPTG was added to cell culture to 1mM IPTG finally, and incubated 3h at 171 rpm, 37℃ in orbital shaking incubator.
SECTION: Sample preparation
3. Preparing several 100mL flasks by filling the flasks with MBBR carrier K1. Autoclave all the flasks and dry them in an oven at 60s℃.
 


SECTION: Sample preparation
4. Cells cultures and silver nitrate (to 6μM) solution were mixed in advance. Add the mixture into each bottle.
 

SECTION: Sample preparation
5. Incubate all bottles in a biochemical incubator at37 °C .

SECTION: Absorbance measurement
6. At the day of measuring the absorbance, take the flasks to be tested out from incubator.
SECTION: Absorbance measurement
7. Using a pair of forceps, carefully pick out the carriers that has not been submerged into the culture and discard.
 

SECTION: Absorbance measurement
8. Randomly pick five carriers out from culturing flask and put into a 50 mL centrifuge tube. Label this tube as "Washing tube".
 

SECTION: Absorbance measurement
9. Wash these five carriers using 1X phosphate-buffered saline (PBS) for three times. Collect all eluents.
 

SECTION: Absorbance measurement
10. Use a dropper, add 2-3 mL 1X PBS into the Washing tube. Cap the tube and rinse every corner of the inside wall of tube using the PBS added by rotating the tube. Collecting the eluents by pouring these PBS into Eluent tube. Repeat these steps for three to five times.

 

SECTION: Absorbance measurement
11. Centrifuge the Eluent tube at10000 rpm, 10 min .

SECTION: Absorbance measurement
12. Discard all supernatants.

 

SECTION: Absorbance measurement
13. Add 3 µL  1X PBS into Eluent tube. Resuspend the pellet.

SECTION: Absorbance measurement
14. Measure the absorbance of the liquid   at OD600. Record the absorbance.

SECTION: Absorbance measurement
15. Repeat steps from Step 7 to Step 14 for three times. Calculate the mean value for the three measurements. A growing curve of biofilm can be generated from the data collected.
TITLE: Digoxigenin Labeled In Vitro Transcription and Ribogreen Quantification
AUTHORS: Allen Institute for Brain Science

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is used for in vitro transcription (IVT) reaction generating DIG-labeled anti-sense (AS) RNA from polymerase chain reaction (PCR) product for in situ hybridization (ISH), as well as fluorescent quantification of products using Ribogreen immediately following.  This protocol is written for 96 reactions but can also be used for single reactions or in multiples of 96 by adjusting the amount of materials and equipment appropriately.</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: Field Genomics Protocols
AUTHORS: Mrinalini Watsa, Gideon Erkenswick, Stefan Prost, Aaron Pomerantz

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This collection of protocols are examples of protocols used for education in high throughput sequencing with Oxford Nanopore Technology's MinION sequencer. </div></div>

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Male adult Sprague–Dawley rats, weighing 270–300 g, obtained from the Animal Center of the Fujian Medical University, were anesthetized with intraperitoneal sodium pentobarbital (50 mg/kg). After intravenous injection of heparinized (50 IU) the hearts were quickly excised and fixed on a Langendorff apparatus (Radnoti, Monrovia, CA, USA) and perfused with 37°C Krebs–Henseleit buffer (KHB) at constant pressure (75 mmHg). After equilibrated with KHB for 15 min, isolated hearts were randomly divided into five groups: 1. Sham group: hearts subjected to 195-min perfusion without I/R injury; 2. I/R group: hearts received 30- min ischemia followed by 120-min reperfusion; 3. SPC group: 2.5% sevoflurane preconditioning was gived for 15 min followed by 15-min washout before ischemia; 4. SPC+MF1 group: combined application of MF1(ImClone, Somerville, NJ, USA), rat anti-VEGFR-1 monoclonal antibody,10 μmol/L and 2.5% sevoflurane preconditioning for 15 min followed by 15-min washout before ischemia; 5. SPC+PIGF group: combined application of PIGF(Abcom, Cambridge, United Kingdom)10 μmol/L and 2.5% sevoflurane preconditioning for 15 min followed by 15-min washout before ischemia.  To implement sevoflurane preconditioning, the KHB was pre-equilibrated with sevoflurane under a separate reservoir with a Vapor 2000 (Dräger Medical AG &amp; Co., Lübeck, Germany) using an air bubbler.</p>

[STEPS]
TITLE: Electrode cleaning solution
AUTHORS: Michael Economo

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">How to make a cleaning solution for cleaning silicon probes</div></div>

[STEPS]
?. Make 1.2X strength cleaning solution (167.67 mL)First, measure  H2O in beakerAdd: NaCl 10% SDS 0.2 M Tris Buffer, pH 8.5
116.67 ml
0.117 g
10 ml
50 ml
?. [On day of use]  Add  10X trypsin to  1.2X cleaning solution to make 12 mL 1x cleaning solution
2 ml
10 ml
TITLE: DOH Workshop Protocol Parts 1, 2 and 3
AUTHORS: Vesa Qarkaxhija, Bryan Wee, Natalie Ring

[DESCRIPTION]
This collection of protocols details the extraction of high-molecular-weight DNA from Gram-negative bacterial culture, purification of dsDNA, removing contaminants and low molecular weight DNA, and creation of Nanopore sequencing libraries for the MinION flow cell.

[STEPS]
TITLE: Error-prone PCR (Random Mutagenesis)
AUTHORS: NUS iGEM

[DESCRIPTION]
2023 NUS-Singapore iGEM Team followed this protocol to introduce random mutation in DNA fragments. 

[STEPS]
SECTION: Error-prone PCR (Mutagenesis)
1. Add the primers, the DNA template, and the following reagents from the  into a PCR tube to make a 50 µL PCR sample:
     DI
  water      41.5μL        10x Mutazyme II (10x Reaction Buffer)      5μL        40mM
  dNTP Mix      1μL        Each Primer (both forward & reverse)      0.5μL        Mutazyme
  II      1μL        Template      0.25μL   

SECTION: Error-prone PCR (Mutagenesis)
2. Mix the solution well.
SECTION: Error-prone PCR (Mutagenesis)
3. Place the sample into the Thermal Cycler and set it with the following conditions:

 

SECTION: Error-prone PCR (Mutagenesis)
4. Upon finishing the PCR steps, add 10 µL of DNA loading dye.

SECTION: Error-prone PCR (Mutagenesis)
5. Proceeds to the gel electrophoresis to isolate the gene fragment of interest.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Denville LE Agarose is an all purpose agarose for routine nucleic acid electrophoresis of fragments between 500bp-23,000 bp.</p>
<p> </p>
<p>Denville LE Agarose has no detectable DNase or RNase activity.</p>

[GUIDELINES]
<p><strong>Introduction</strong></p>
<p>Denville LE Agarose is an all purpose agarose for routine nucleic acid electrophoresis of fragments between 500bp-23,000 bp. Denville LE Agarose has no detectable DNase or RNase activity.</p>
<p> </p>
<p><strong> Analytical Specifications</strong></p>
<p>Gelling temperature (1.5%)                     36°C ±1.5°C</p>
<p>Melting temperature (1.5%)                   &gt;90°C</p>
<p>Gel strength (1%)                                 &gt;1,200 g/cm²</p>
<p> </p>
<p><strong>Applications </strong></p>
<p><br /> Analytical electrophoresis of DNA and RNA &gt;1,000 bp</p>
<p> Blotting of DNA and RNA </p>
<p> </p>
<p><strong>Suggested Agarose Concentrations</strong></p>
<p> </p>
<p><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/ha2edne.png" data-src="https://s3.amazonaws.com/pr-journal/ha2edne.png" data-ofn="Screen Shot 2016-12-15 at 11.28.02 AM.png" /><br /> </p>
<p><strong>Dye Mobility Table</strong></p>
<p>Migration of double-stranded DNA in relation to Bromophenol Blue (BPB) and Xylene Cyanol (XC) in Denville LE Agarose Gels.</p>
<p><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/ha3edne.png" data-src="https://s3.amazonaws.com/pr-journal/ha3edne.png" data-ofn="Screen Shot 2016-12-15 at 11.28.49 AM.png" /> </p>
<p> </p>
<p><br /><br /></p>

[STEPS]
?. 
?. 
?. 
?. 
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?. 
?. 
?. 
?. 
?. 
?.
TITLE: OMS Atlas FFPE Spatial Mapping
AUTHORS: Brett   Johnson, Danielle Galipeau, George Thomas

[DESCRIPTION]
This protocol describes the procedure by which the OMS Atlas serially sections a FFPE block, prepares the resulting slides, and then distributes the specimens for downstream analysis.

[BEFORE_START]
Transfer FFPE blocks to OHSU Knight Histopathology Shared Resource (HSR) for sectioning and processing. Coordinate execution of this sectioning protocol to allow for shipping within the same business week.

[STEPS]
SECTION: Preparation
1. Verify the identity of the FFPE block to be cut against written request for sectioning.
SECTION: Preparation
2. Label all slides with a unique BEMS ID and slide number, corresponding to the written request and FFPE spatial map (below).
 

SECTION: Sectioning
3.  Align block on microtome to minimize tissue loss.
SECTION: Sectioning
4. Face into block at 5μm until full section of tissue is achieved.
SECTION: Sectioning
5. Cut adequate ribbon at 5μm to cover all 8 serial sections.
SECTION: Sectioning
6.  Mount tissue sections onto Tanner slides to maintain serial order and orientation of sections.
SECTION: Processing
7.  Perform hematoxylin and eosin (H&E) staining on slides #1 and #8.
8. Bake slides #2, 3, 5, and 6.

8.1. Place slides #2, 3, 5, and 6 in 55 °C oven overnight.
8.2.  The next morning, raise the oven temperature to 65 °C for up to 60 min.
Note: Slides should be baked at 65 °C for at least 30 minutes.

8.3.  Remove slides from oven. Place slides in slide boxes and store at room temperature.
9. Deliver slides #2 through #7 to BioLibrary for distribution.
TITLE: DNA Gel Electrophoresis
AUTHORS: An.Huang

[DESCRIPTION]
Agarose gel electrophoresis can help determine the mass of a certain DNA fragment. This protocol will help user conduct gel electrophoresis appropriately.

[STEPS]
1. Mix 1 g agarose with 100 mL electrophoresis buffer (1X TAE) in a flask, then heat and stir until agarose is completely dissolved.
 

4. Add 10 µL 10000X GelRed nucleic acid stain into molten agarose and mix well.

2. Wait until the molten agarose cools down to around 40 °C . 

 



3. While waiting for the cool down of molten agarose, set up and level the gel casting tray and the gel casting platform on a flat area. Inset the comb.
5. Pour the molten agarose onto the tray. Immediately rinse out the flask before any remaining agarose sets in it.
6. Wait at least 20 min for gel solidification.


7. Once the gel has completely solidified, gently remove the comb by pulling it up slowly and vertically.
8. Carefully place the tray in the gel tank. Add electrophoresis buffer (1X TAE) until covering the whole gel surface.
6.1. While waiting for gel solidification, prepare the samples as follows:

Mixing the components:
6X gel loading dye                     X μL
DNA sample/DNA ladder       5X μL
 

9. Pipette the prepared samples using a P20 (2μL-20μL) pipette to the wells.
 


10. Run the gels at 90 V, approximately 50 mA, for 90 min.
11. When the run is finished, carefully take the gel tray with gel out from gel tank. Photograph the gel under ultraviolet (UV) light.
TITLE: Plasma protein expression pattern in congenital analbuminemia – systematic review protocol
AUTHORS: Bailey M. Foster, Afsoun Abdollahi, Gregory C. Henderson

[DESCRIPTION]
Congenital analbuminemia patients do not express albumin due to genetic mutation.  Here a protocol is described for carrying out a systematic review of published case reports on congenital analbuminemia patients. The goal is to assess abundances of a variety of plasma proteins and total plasma protein concentration in this patient population. From the literature search, concentration of individual plasma proteins, plasma protein classes, and total plasma protein concentration is assessed in comparison to reference ranges. 

[STEPS]
SECTION: Article selection
1. Literature search is carried out using multiple databases for case reports on congenital analbuminemia patients that were written in the English language.
SECTION: Article selection
2. Case reports on congenital analbuminemia patients are included in the systematic review when the genetic condition was confirmed or could be reasonably inferred, and if plasma proteins were measured.
SECTION: Article selection
3. Exclusion of papers is carried out when patients were diagnosed with another acute or chronic disease or illness, except conditions expected to be reasonably common in the overall human population (obesity, hypercholesterolemia). Reports of patients with edema would not be excluded because edema could be directly related to congenital analbuminemia.
SECTION: Protein Selection from the included articles
4. Total plasma protein concentrations are included in the analysis.
SECTION: Protein Selection from the included articles
5. Individual proteins or protein classes are included in the analysis if reported in at least 3 patients of the same sex. 
SECTION: Protein Selection from the included articles
6. It is ascertained if the protein abundance is above, within, or below the clinical reference range.
SECTION: Protein Selection from the included articles
7. The relative frequency of a protein abundance being above or below the reference range is used to determine the strength of the association with congenital analbuminemia .
TITLE: Integrative taxonomy of Ophiothrix (Echinodermata: Ophiuroidea)
AUTHORS: Renata Alitto, Michela Borges, Letícia Dias de Oliveira, Helena Serrano

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">In this protocol, we highlight that molecular data did not have preference over morphological features.</div></div>

[STEPS]
?. [Character sets]
Species boundaries among populations of Ophiothrix were evaluated using four independent character sets in order to test: i) external morphology, ii) arm microstructures morphology, iii) morphometry, and iv) molecular data (16S and COI fragments).
?. [Congruence framework]
The CS were classified according to the congruence framework of Padial et al. (2010), when there was concordance of, at least, three data sets analyzed.
TITLE: U Mass - Body composition (whole body)
AUTHORS: Jason Kim

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary:</span><span style = "font-weight:bold;"> </span></div><div class = "text-block">
The EchoMRI 3-in-1 uses ¹H- magnetic resonance spectroscopy to noninvasively measure whole body fat mass, lean mass, and water mass in awake mice. Whole body fat mass is a measure of adiposity that is altered in obesity. Whole body lean mass may be altered by exercise and in mouse models of altered metabolism.</div></div>

[STEPS]
?. Place awake mice into an instrument column.
?. Measure whole body fat mass, lean mass, and water mass using instrument standard operating procedure.
TITLE: Make serotonin and naloxone drug plates
AUTHORS: Serena Ding

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Protocol for making a set of three serotonin drug plates and a set of three naloxone drug plates. Serotonin (20 mM) is added to molten agar, whereas naloxone (10 mM) is made into a 10X stock solution in water first before being spread over agar plates to achieve the final concentration. Each 35 mm drug plate contains 3 mL low peptone NGM agar and the drug, and is always made up fresh the day before imaging experiments. </div><div class = "text-block"><span>For naloxone methods, refer to </span><a href="https://www.ncbi.nlm.nih.gov/pubmed/25898004" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://www.ncbi.nlm.nih.gov/pubmed/25898004</span></a><span>. </span></div></div>

[STEPS]
?. First identify the drugs (serotonin and naloxone) to be used in the study and ensure that they are correctly labelled and handled.
?. Since these are powered compounds, calculate the desired weight required for 10 mL of serotonin agar (at 20 mM final concentration), and for 1 mL of 10X naloxone stock solution (10X is 100 mM, for 10 mM final concentration). These volumes will give 3 drug plates each.
?. Inside a fume hood, with an analytical balance weigh out the desired amount of serotonin and leave aside. Weigh out the desired amount of naloxone and transfer the powder into a 1.5 mL Eppendorf tube labelled with the compound and the final concentration. To the naloxone tube, add 1 mL sterile water to make up the 10X stock solution, and close the lid of the Eppendorf tube.
?. Take the naloxone stock solution tube out of the fume hood. To ensure the compound is dissolved fully, vortex the Eppendorf tubes (ensuring the lid is firmly in place) at setting 10 for 30 seconds.
?. Make up 500 mL low peptone NGM agar and put it on autoclave.
Do not attempt to use a smaller volume even though not much agar is required for drug plate preparation, as chemical weights will be too small to be measured accurately.
?. When the autoclave cycle is done, add salts as normally required.
?. Remove 10 mL of molten low peptone NGM agar, take it to the fume hood and add the pre-weighed serotonin to achieve 20 mM final concentration. Mix well. Dispense 3 mL into each of three pre-labelled plates using a 10 mL serological pipette. Let serotonin plates set.
?. Using a different 10 mL serological pipette, draw up 10 mL of molten agar (without serotonin) and dispense 3 mL each into a different set of three pre-labelled plates. Let the plates set (these will become the naloxone plates).
?. Wrap drug plates in foil and leave them overnight at 4°C to let the drugs diffuse. Use for experiments immediately the next day.
?. Add 300 μL of 10X naloxone stock solution on top of each plate from the last step. Let these plates dry in a laminar flow hood for 3 hours.
?. Get six 35 mm petri dishes and label three as serotonin (20 mM) plates and three as naloxone (10 mM) plates.