eamag/protocols-with-steps huggingface dataset:
TITLE: HTMCs 3D culture method using Corning Matrigel Matrix
AUTHORS: Stefania Vernazza

[STEPS]
?. [Cell Cultures]
Human trabecular meshwork cells (HTMC) and their Growth Medium with FBS, theTrabecular Meshwork Growth Medium (TMGM), from Cell APPLICATION INC.  Maintain the HTMCs at 37◦C in a humidified atmosphere containing 5% CO2, according to the manufacturer’s indications. Sub-cultured HTMCs by TripLETM Express (Invitrogen Life Technologies) treatment when the original flask is approximately 75% confluent.
?. [Cells embedded]
Slightly centrifuge 2.5 x 10 5/cm3 cells for 5 min at 90 rcf, descard the supernatant and gentle resuspend the cell pellet in undiluted Matrigel™ at 4°C, following the application of this formula: 
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							Volume of added Matrigel (ml)/culture chamber growth area (cm2).i.e.: 0.2 ml /[(1.77/2)2 3.14 ] = 0.113 cm --> 1.13 mm thickness/growth area.
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							Gently transfer the embedded HTMCs by pipette into culture chambers and add the culture medium after 15 min, which is the necessary time for the Matrigel to reach its gelling state. Then, incubate each culture chamber under standard culture conditions.
?. [Experimental treatment]
Before performing experimental treatments, in order to reduce any Fetal Bovine Serum (FBS) interference on cellular proliferation,  maintain the 3D HTMC culturesfor at least 24 hrs in low and high glucose DMEM (1:1 mix), 2mM L-glutamine, 0.5% gentamicin and 100μg/ml streptomycin, w/o (FBS), according to Keller et al., (2018 ) Experimental Eye Research. 2018;171: 164–173. doi:10.1016/j.exer.2018.03.001. Then, proceed with experimental conditions.
?. [3D HTMCs recovery]
Release 3D-HTMCs from Matrigel at the end of experimental treatments  with Corning® Cell Recovery Solution, following the "RECOVERY OF CELLS FROM CORNING MATRIGEL MATRIX" protocol, providing by Corning. Then, process the samples for further molecular analysis.
TITLE: dSTORM of actin in fixed HeLa cells
AUTHORS: Ezra Bruggeman, ruby peters

[DESCRIPTION]
This is a protocol for the preparation of a HeLa DTS cells for dSTORM imaging of the actin network. This protocol was used to generate the data shown in Figure 4 of the following publication:

Bruggeman et al., POLCAM: Instant molecular orientation microscopy for the life sciences. bioRxiv 2023.02.07.527479 (Feb 2023), doi: https://doi.org/10.1101/2023.02.07.527479

[STEPS]
SECTION: Cell culture
1. HeLa TDS cells were cultured in DMEM (Gibco, Invitrogen) supplemented with 10 % Fetal Bovine Serum (FBS, Life Technologies), 1 % penicillin/streptomycin (Life Technologies), and 1 % glutamine (Life Technologies) at 37 °C+ 5 % CO2.

SECTION: Cell culture
2. Cells were periodically tested for mycoplasma contamination and passaged 3 times per week.
SECTION: Cell culture
3. Cells were plated at low density on high-precision glass coverslips (MatTek, P35G-0.170-14-C) 1 day prior to fixation for dSTORM experiments.
SECTION: Fluorescent labelling
4. Simultaneously fix and permeabilize the cells in cytoskeleton buffer (CBS, 10 mM MES, 138 mM KCl, 3 mM MgCl2, 2 mM EGTA, 4.5 % sucrose w/v, pH 7.4) + 4 % paraformaldehyde (PFA) and 0.2 % Triton for 6 min at 37 °C

SECTION: Fluorescent labelling
6. Wash the cells 3 times in PBST (PBS supplemented with 0.1 % Tween).
SECTION: Fluorescent labelling
8. Wash the cells 3 times in PBST.
SECTION: Fluorescent labelling
11. Incubate the cells with Alexa Fluor‱ 488 Phalloidin (A12379, Invitrogen, 1:50 in PBS) for 60 min in the dark.

SECTION: Fluorescent labelling
13. Prior to dSTORM imaging, replace the PBS with dSTORM imaging buffer (base buffer consisting of 0.56 M glucose, 50 mM Tris (pH 8.5), and 10 mM NaCl supplemented with 5 U/mL pyranose oxidase (Sigma, P4234), 10 mM cysteamine (Sigma, 30070), 40 µg/mL catalase (Sigma, C100) and 2 mM cyclooctatetraene (Sigma, 138924).
SECTION: Fluorescent labelling
5. Further fix the cells in CBS + 4 % PFA for 14 Mass Percent at 37 °C.
SECTION: Fluorescent labelling
7. Permeabilize a second time in PBS + 0.5 % Triton for 5 min at Room temperature.

SECTION: Fluorescent labelling
9. Blocked the cells for 30 min in 5 % BSA.

SECTION: Fluorescent labelling
10. Wash the cells 3 times in PBST.
SECTION: Fluorescent labelling
12. Wash the cells 3 times in PBS.
TITLE: NEXT Gel - CHEM 584 
AUTHORS: Ken Christensen

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">General Information:</span><span> VWR Life Science AMRESCO’s NEXT GEL® products for denaturing gel electrophoresis are proprietary, ready-to-pour solutions comprised of acrylamide, bis-acrylamide, gel buffer, and SDS. The unique chemistry of NEXT GEL® eliminates the need for a stacking gel, thus reducing gel preparation time and extending the separation matrix available for electrophoresis, enabling the resolution of small peptides and high molecular weight proteins in the same gel.</span></div><div class = "text-block">NEXT GEL® solutions polymerize upon addition of ammonium persulfate and TEMED and are fully compatible with all standard electrophoresis equipment, SDS-PAGE staining procedures, and downstream applications including 2D electrophoresis, western blot, transfer, protein sequencing, and MALDI analysis. Each NEXT GEL® acrylamide solution is supplied with NEXT GEL® Running Buffer, 20X, which is essential for optimal gel performance.</div></div>

[STEPS]
?. Add  of TEMED and  of freshly made 10% w/v ammonium persulfate (APS) to  ofNEXT Gel solution in a conical tube. Tighten cap and mix immediately by gently inverting. Pour between prepared glass plates, filling to the top.
3 µl
30 µl
5 mL
?. Insert an appropriate comb and allow gel to polymerize for up to   minutes.No stacking gel required!
?. Remove comb. Rinse wells with water to remove any residual gel pieces.
?. Assemble mini-gel system. Dilute NEXT GEL® Running Buffer, 20X to 1X by diluting 1:20 in deionized water. Prepare sufficient 1x NEXT Gel Running Buffer from a20X stock solution to fill both the anode and cathode chambers. For the Bio-Rad Mini-Gel Tetra System gel apparatus that we use in the lab, this means that you will need  of 1x Running Buffer for 1 gel and  of Running Buffer for 2 gels.
350 mL
700 mL
?. Prepare molecular weight markers and samples per standard preparation procedures andload gel wells. Use the 6x SDS Loading Buffer for preparing samples.
?. Run the gel at 150 volts for up to  or until dye reachesbottom of the gel.
?. Remove and stain gel for proteins using the Coomassie Blue staining solution for up to  or overnight. Transfer to nitrocellulose if performing a western blot.
?. For Coomassie Blue stained gels, destain using the Destain solution for up to 1h or to overnight. The addition of a Kimwipe to Destain can enhance the destaining process. Be careful not to destain too long as the protein bands will lighten.
?. Prepare a fresh solution of 10% w/v ammonium persulfate in water. 1 mL is sufficient for many gels.
?. Disassemble the gel apparatus and proceed with the downstream application.
?. [FAQ's]
TITLE: GATK Nuclear variant discovery and consensus assembly
AUTHORS: Graham Etherington

[DESCRIPTION]
The European polecat (Mustela putorius) is a mammalian predator which breeds across much of Europe east to central Asia. In Great Britain, following years of persecution the European polecat has recently undergone a population increase due to legal protection and its range now overlaps that of feral domestic ferrets (Mustela putorius furo). During this range expansion, European polecats hybridised with feral domestic ferrets producing viable offspring. Here we carry out population-level whole genome sequencing on domestic ferrets, British European polecats, and European polecats from the European mainland and find high degrees of genome introgression in British polecats outside their previous stronghold, even in those individuals phenotyped as ‘pure’ polecats. 

[STEPS]
SECTION: BWA mapping
1. Here we map our reads to the domestic ferret genome, run the GATK pipeline to identify variants.

SECTION: BWA mapping
1.1. Map the reads to the reference and sort the output

 

2. 
GATK Pipeline
SECTION: GATK pipeline
2.1. Mark Duplicates, add read groups, call variants, calculate GATK parameters, filter variants, create base quality recalibration table, apply it, and re-run variant calling.
The GenomeHelper program can be found here:
https://github.com/ethering/GenomeHelper
The mean value of SNP quality, read depth, and mapping quality (QUAL, DP, and MQ values respectively from the VCF file) were calculated using GenomeHelper, 



 

SECTION: GATK pipeline
2.2. Identify SNPs

Next, we want to create a GATK GenomicsDB and then joint genotype all of the samples to get a multi-sample VCF file of SNPs

Firstly, In our 'vcfs' directory, we need a tab-delimited sample map, linking the sample-specifice GVCF file to a sample name, and an intervals file.

cohort.sample_map
 


We also need to to create an intervals file, which will just be a list of scaffolds in the reference sequence
 

SECTION: GATK pipeline
2.4. Create a consensus genome sequence for each sample

gatk_farm.sh
 

Note 1. 
FastaAlternateReferenceMaker puts an incremented number after the ">" and then the co-ordinates of the sequence after the scaffold name, so:

>Super-Scaffold_5
becomes
>5 Super-Scaffold_5:1-33914829

I use the following sed at the end of the script to go back to the original fasta header:

 

As a helper script, if using SLURM, you can use the following script to submit each job.

 

SECTION: GATK pipeline
2.3. Create the GenomicsDB, Genotype each sample and select only SNPs
TITLE: u-Smell-it olfactory test  
AUTHORS: dkt 

[STEPS]
?. Receive Olfactory/Chemosensory test CARD
?. Scan QR code on the Olfactory Card (using any smart phone) and the camera function (they automatically recognize QR codes)(Note: For people without access to a smart-phone there will be an automated call-in center that will provide voice prompts for choices of scent options and will connect to the centeralized server, similar to the Apps)
?. Dowload 'u-Smell-it' App on iOS (Apple) or Android phone
?. Scan the QR-Code on the Card in the AppNote: After downloading the software Steps 1-3 are not necessary for additional tests (as would usually happen in repeated testing)
?. Follow the App instructions
?. Rub or scratch window #1 with finger(nail)
?. In app choose the best of 4 scent options (one of which is the true scent) or pick the "no scent" option
?. Repeat 5.1 and 5.2 for all 5 scent window
?. Respond to symptom self-reporting questions
?. See the olfactory score follow the advice
?. NEXT day, or at specified interval repeat with a new card
TITLE: Blackwater medium
AUTHORS: Callen Hyland, Rob Steele

[DESCRIPTION]
Blackwater medium is a laboratory growth medium for Hydra that replicates the chemical composition of blackwater swamps. This medium has lower low ionic strength than traditional Hydra medium and contains humic acid. Blackwater medium is ideal for Hydra species isolated from blackwater swamps and improves the health of some transgenic Hydra lines that are prone to infection.

[GUIDELINES]
Please see Low cost methods for Hydra care for general guidelines for working with Hydra.

[STEPS]
SECTION: Stock solutions
1. Make stock solutions in 50 mL conical tubes with DI water.

 

SECTION: Stock solutions
2. Not all of the humic acid sodium salt will go into solution. Filter the humic acid solution with a syringe filter or vacuum filter to remove undissolved particles.
SECTION: Working solution
3. Prepare blackwater medium by diluting stock solutions in DI water. Use a 1 L bottle, or scale up volumes for a large plastic carboy. Mix well before using.
TITLE: Derma PGX Cream Review (Clinically Tested) Natural Skin
AUTHORS: Derma PGX Cream

[DESCRIPTION]
Derma PGX Anti Aging Cream (Clinically Tested) Natural Skin Look Younger! Review, Benefits, Result, and Cost!
Revitalize Your Skin Today With Derma PGX Cream
Derma PGX Review[Organic Skin Care]More Useful Than Other Products!

 


[STEPS]
TITLE: ATR FTIR spectoscopy of aqueous cell culture
AUTHORS: Emrulla Spahiu, Senol Dogan, Jörg Schnauß, Mayda Gursel, Feride Severcan

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">ATR-FTIR Spectroscopy of aqueous cell culture samples is explained step-by-step. Infrared spectra acquisition, processing and analysis is included briefly.</div></div>

[STEPS]
?. [Spectra acquisition]
Clean the ATR crystal using a cellulose wipe and water-ethanol-water sequence. Scratching the crystal surface must be avoided.
?. [Spectra acquisition]
Leave the crystal to air-dry
?. [Spectra acquisition]
Arrange the temperature of the working environment to
25 °C
?. [Spectra acquisition]
Select “Scan and Instrument Setup” and input sample name, scan range (650-4000 cm-1), scan number (128), and  spectral resolution (2 cm-1). This step needs optimization for specific sample being used.
?. [Spectra acquisition]
Scan the backgroung spectrum to overcome the probable atmospheric interference that can be triggered by H2O and CO2 molecules present in the air, subtracted automatically by the software.
?. [Spectra acquisition]
Additionally, record PBS as sample (subtract this manually from each spectrum)This eliminates interference of PBS and flattens the water band located around 2125 cm -1
?. [Spectra acquisition]
Put   cell samples and let to dry with a mild N2 flux for  . This allows cells to settle on the crystal.
1 µl
?. [Spectra acquisition]
Scan the sample. Process takes about  to finish.As the scanning process goes on, one must be sure that the quality check sign is everytime green.
?. [Spectra acquisition]
Scan each sample under the same conditions in three independent scans (the average constitutes a replicate n). Replicates (n=5) are used in data analysis. This minimizes intra-sample variability and eliminates possible variation that might arise from experimental conditions.
?. [Spectral pre-processing]
The avarage of triplicates is taken manually.
?. [Spectral pre-processing]
13-point Savitzky-Golay smoothing
?. [Spectral pre-processing]
Baseline correction at 3800-2750-1800-900 cm-1points.
?. [Principal Component Analysis (PCA)]
Import spectra as data file into
?. [Principal Component Analysis (PCA)]
Define the data sets to 3000-650 cm-1region.
?. [Principal Component Analysis (PCA)]
Make the PCA Model.Define the regions with high variation using the Loadings plots.Check how the replicates are grouping and if there are outliers in Scores plots.
?. [Spectral Calculations]
Peak labelling can be done automaticaly. For increased precision we measured manually:Measure the center of weight of each band. Read the frequency.Compare the peaks you obtained, to the literature:Make an excel file of your peaks and their assignment to the functional groups. For side-peak band assignment, the second derivative spectrum is used.
?. [Spectral Calculations]
Concentration studies are carried out by integrating the curves under the peaks.
?. [Spectral Calculations]
For conformational alteration studies, bandwidth measurements are done manually using the 75% height of the peaks.
TITLE: Detection of anti- Keyhole limpet hemocynin (anti-KLH) in rats by double immunodiffusion (Ouchterlony) technique.
AUTHORS: Angel  Justiz-Vaillant, Norma McFarlane-Anderson

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Keyhole limpet hemocyanin (KLH) is a cooper-containing protein comprising of subunits with MW of 400 kDa. This protein is found in the hemolymph of the sea mollusk Megathura crenulata. It has the ability to enhance the host’s immune response by interacting with monocytes, T cells and macrophages. KLH has been used primarily as a carrier for vaccines and antigens [1]. It was found that chicken immunized with KLH bound peptide raised an anti-KLH immunoresponse [2]. This can be tested by a single method such as the Ouchterlony technique.</div><div class = "text-block">Reference</div><div class = "text-block"><span>1. Aarntzen EH, de Vries IJ, Göertz JH, et al. Humoral anti-KLH responses in cancer patients treated with dendritic cell-based immunotherapy are dictated by different vaccination parameters.</span><span style = "font-style:italic;">Cancer Immunol Immunother</span><span>. 2012;61(11):2003-2011. doi:10.1007/s00262-012-1263-z</span></div><div class = "text-block">2. Justiz Vaillant AA, Anderson MF, Smikle M, Wisdom B, Mohammed W, et al. (2013) Development of Anti HIV Gp120 and HIV Gp41 Peptide Vaccines. J Vaccines Vaccin 4: 206. doi: 10.4172/2157-7560.1000206 </div></div>

[STEPS]
?. Detection of anti-keyhole limpet hemocynin antibodies in rats by double immunodiffusion is carried out.
?. Briefly, 1% agarose gels are prepared and wells cut into the gel using a template.
?. Initially, aliquots of 25 µl each of KLH in concentration of 1 mg/ml are applied to the centre well.
?. The peripheral wells are filled with 25 µl of rat serum post-immunized with an anti-HIV gp120 vaccine.
?. The gels are incubated at RT for 48−72 hours.
?. After that the gels are examined for precipitin lines.
?. An anti-KLH developed in chickens is included as positive control and turtle serum as a negative control.
?. The positive results are taken as the presence of precipitin line/s and negative results, the absence of precipitin lines.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Protocol for separating total RNA under denaturing conditions using Urea-PAGE using the 10 mL Hoefer SE260 <em>Mighty Small</em> system.</p>

[BEFORE_START]
<p>Thoroughly clean all components with ethanol and RNase away.</p>
<p>Heat block to 95˚C.</p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: Gus staining
AUTHORS: lilyli

[DESCRIPTION]
This is a protocol for gus staining. 

[STEPS]
1. 1: Fix with 90% acetone for 20 min;
2. 2: Add 1 mL GUS lotion to wash off acetone, and wash twice:
3. 3: Add GUS dye solution and vacuum on ice for 15-20 min;
4. 4: Place at 37C and observe after a while -- next, after dyeing, proceed to the following steps:
5. 5: Suck out the dye solution, add 1mL 70% ethanol, and stop the dyeing reaction and decolorization:
6. 6: Replace ethanol several times until complete decolorization;
TITLE: Adapter ligation with AMX
AUTHORS: Josh Quick

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This is a subprotocol for generating a library from a single amplicon pool</div></div>

[STEPS]
?. Set up the following AMX adapter ligation reaction:Component                                                                    VolumeEnd-repaired amplicon pools                                      Ligation Buffer (LNB)                                                   Adapter Mix (AMX)                                                       Quick T4 DNA Ligase                                                   Total
30 µl
10 µl
5 µl
5 µl
50 µl
There will be some variation in clean-up efficiencies but expect to carry around 80% through a clean-up.
?. Incubate at room temperature for
?. Add (1:1) of SPRI beads to the sample tube and mix gently by either flicking or pipetting.
50 µl
Vortex SPRI beads thoroughly before use to ensure they are well resuspended, the solution should be a homogenous brown colour.
?. Pulse centrifuge to collect all liquid at the bottom of the tube.
?. Incubate for  at room temperature.
?. Place on magnetic rack and incubate for  or until the beads have pelleted and the supernatant is completely clear.
?. Carefully remove and discard the supernatant, being careful not to touch the bead pellet.
?. Add  SFB and resuspend beads completely by pipette mixing.
250 µl
SFB will remove excess adapter without damaging the adapter-protein complexes. Do not use 70% ethanol as in early clean-ups.
?. Pulse centrifuge to collect all liquid at the bottom of the tube.
?. Remove supernatant and discard.
?. Repeat steps 14-16 to perform a second SFB wash.
?. Pulse centrifuge and remove any residual SFB.
You do not need to allow to air dry with SFB washes.
?. Add EB and resuspend beads by pipette mixing.
15 µl
?. Incubate at room temperature for .
?. Place on magnetic rack.
?. Transfer final library to a new 1.5mL Eppendorf tube.
TITLE: In vitro α-amylase and α-glucosidase inhibitory assay
AUTHORS: Maira Rubí  Segura-Campos

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Diabetes mellitus is an endocrine disease characterized by hyperglycemia that affects the metabolism of carbohydrates, proteins, and fats. The unique approach to decreasing postprandial hyperglycemia is to reduce or slow down dietary carbohydrate digestion. The inhibition of α-amylase and α-glucosidase enzymes is a strong therapeutic goal of controlling the postprandial glycemic reaction. This protocol describes the anti-hyperglycemic activity of aqueous extracts measuring the in vitro inhibition of these enzymes.</div></div>

[STEPS]
?. [In vitro α-amylase inhibitory assay: Preparation of the substrate solution]
Starch suspended in 0.2 mL of 0.5 M Tris-HCl pH 6.9 (0.01M CaCl2)
[Starch]
?. [In vitro α-amylase inhibitory assay: Pre-incubation]
Boil (5 min) and pre-incubate the substrate solution
?. [In vitro α-amylase inhibitory assay ]
Aqueous extract dissolution
[dissolved with 1 mL of 0.1% DMSO]
?. [In vitro α-amylase inhibitory assay: Mix and incubation ]
Mix 0.2mL of the aqueous extract with the substrate solution, and 0.1 mL of porcine pancreatic amylase in Tris-HCl buffer (2 U/mL)
?. [In vitro α-amylase inhibitory assay: Stop reaction]
Add 0.5 mL of acetic acid (50% v/v) and centrifuge
[at 1811 g]
?. [In vitro α-amylase inhibitory assay: Calculation of the α-amylase inhibition]
The inhibitory activity is calculated using the formula (Ac+) – (Ac-) – (As - Ab)/(Ac+) – (Ac-) × 100, where Ac+, Ac-, As, Ab are defined as the absorbance, at 595 nm,  of 100% enzyme activity (only solvent with enzyme), 0% enzyme activity (only solvent without enzyme), the test sample (with enzyme), and a blank (a test sample without enzyme), respectively
?. [In vitro α-glucosidase inhibitory assay: Mix and incubation]
Mix 2 U/mL of the α-glucosidase enzyme with 20 μL of aqueous extract (1mg/mL)
?. [In vitro α-glucosidase inhibitory assay: Initiation of reaction and incubation]
Add 1 mM of pNPG (pH 6.8; buffered with 50mM of phosphate) and incubate
20 µl
?. [In vitro α-glucosidase inhibitory assay: Stop reaction ]
Add 1 mM of sodium carbonate
50 µl
?. [In vitro α-glucosidase inhibitory assay: Calculation of the α-glucosidase inhibition]
The inhibitory activity is calculated using the formula (Ac+) – (Ac-) – (As - Ab)/(Ac+) – (Ac-)× 100, where Ac+, Ac-, As, and Ab are defined as the absorbance, at 405 nm, of 100% enzyme activity (only solvent with enzyme), 0% enzyme activity (only solvent without enzyme), the test sample (with enzyme), and a blank (a test sample without enzyme), respectively.
Acarbose is an anti-hyperglycemic drug that is used as a control
TITLE: Viral PEG purification
AUTHORS: sarah.schulz

[DESCRIPTION]
Protocol for viral particle purification via PEG percipitation

[STEPS]
1. Add 30 ml of ice cold PBS to 3 g of faecal sample and mix by vortexing at high speed for 5 min, then keep on ice
2. Centrifuge sample at 5000 rpm for 10 min at 4°C and transfer the supernatant to a clean 50 ml centrifuge tube
3. Filter the supernatant through a 0.45 μm syringe filter
4. Add 10 % 10% w/v PEG-8000 in saline (0.5 M NaCl) to filtrate, mix by inverting and incubate at 4°C overnight
5. Centrifuge sample at 5000 rpm for 20 min at 4°C and discard the supernatant. 
6. Re-suspend pellet in 200 μl PBS and transfer it to a 1.5ml Eppendorf tube
7. Add an equal volume of chloroform to the sample and mix gently before centrifuging it at 2500 rpm for 5 min at room temperature
8. Transfer the aqueous phase into a new and sterile 1.5 ml tube
9. Add 40 μl of 10 mM CaCl2 and 50 mM MgCl2 to the sample, as well as 10 U of DNase and 10 U of RNase A
10. Incubate samples at 37°C for 2 h and then place them in a heatblock at 70°C for 10 min
11. Store at 4°C