eamag/protocols-with-steps huggingface dataset:
TITLE: ONT DNA Barcoding Fungal Amplicons w/ MinION &amp; Flongle
AUTHORS: Stephen       Douglas Russell

[DESCRIPTION]
This collection of protocols outline a working methodology to DNA barcode fungal specimens. This process will work for dried tissue, fresh tissue, or with DNA template that has already gone through an alternative extraction protocol. It takes you from computer/software setup through DNA extraction, amplification, library preparation, sequencing, and primary data analysis.

This particular workflow is designed for use with Flongle 9.4.1 flowcells and V12 Ligation chemistry.


[STEPS]
TITLE: Protocol for manual von Frey
AUTHORS: Nathan Rizo, Lauren Smith, Olivier George

[STEPS]
?. [Procecdure]
Place netting on surface set: (x2)
?. [Procecdure]
Place clear boxes on top of netting (4 boxes x2 = 8)
?. [Procecdure]
Place one animal per box, leave animals in box for 10 minutes to stabilize to the environment
?. [Procecdure]
Label your experimental sheet (L/R, X/), Name/Date/Experiment/Rats)
?. [Procecdure]
Choose your starting filament handle (example: 4.93)
?. [Procecdure]
Measure the left paw then the right pawa. The animal is facing away from you - the left paws your leftb. Use only the hind pawsc. Place the needle in the center of the pawd. Make sure the needle goes through the center of the grid:
?. [Procecdure]
Apply the filament just long enough for the animal to respond (1-2 seconds)
?. [Procecdure]
Record an X for no response or an 0 for responsea. A response is the animal withdrawing the paw upon applying the filamentb. Start at the BOTTOM LEFT of the cell on the record sheet for left paw (bottom right for right paw)
?. [Procecdure]
IF NO RESPONSE - move down to the next cell, increasing the handle # (example: 5.07) and repeat steps 6-8a.   AB14.93X25.07
AB14.93X25.07
?. [Procecdure]
IF RESPONSE - move up to the previous cell, decreasing the handle # (example: 4.93)a.   AB14.93X25.070
AB14.93X25.070
?. [Procecdure]
Keep going until you have two non-responses followed by two responses in sequential cellsa.   AB14.93XX25.0700
AB14.93XX25.0700
?. [Procecdure]
Once you have two non-responses followed by two responses in sequential cells for both left and right paws that animal is done, move on to the next animals
?. [Procecdure]
Once all animals are done, return them to their home cage and clean the net and work surface
?. [Procecdure]
Return all CLEANED equipment where you found it
TITLE: Cyanobacterial OD based Growth Assay - iGEM IISER Pune 2021
AUTHORS: misaal.bedi 

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol can be used to measure the optical density (OD) based growth of Cyanobacteria to obtain a growth curve.</div></div>

[STEPS]
?. [Sample Preparation]
Start with a single colony of Synechococcus elongatus UTEX 2973 and inoculate it in  of liquid BG-11 media containing  of NaCl.
10 mL
?. [Sample Preparation]
Allow the colony to grow to a dark green color for 1-2 days in a shaker incubator at 150 rpm, at the temperature for which you want a growth curve.We are using the Thermo Scientific MaxQ 6000 shaking incubator.
?. [Sample Preparation]
Transfer the growing mixture into  of liquid BG-11 media containing  of NaCl.
90 mL
?. [Sample Preparation]
Allow the  the growing mixture to grow for 24 hours
100 mL
?. [Sample Preparation]
Take  of the culture and measure its optical density in a spectrophotometer at a wavelength of 730 nm, since it lies outside of the absorption spectrum of cyanobacterial pigments (Blankenship et al., 2011), after calibrating the spectrophotometer to the BG-11 media with 150 mM NaCl. We are using the Eppendorf Biospectrometer Basic spectrophotometer.
1 mL
?. [Sample Preparation]
If the OD of the culture is of a high-density (OD= 0.8-1) proceed with the next step. If not, allow the culture to grow for some more time before taking an OD measurement again.If the OD of this culture is >1, subculture   of the culture into   of the BG-11 media with   NaCl and allow it to grow again before taking an OD measurement.
10 mL
90 mL
?. [Sample Preparation]
After obtaining the OD from the previous step, use the formula O1.V1 = O2.V2 (where O is OD and V is the volume of the culture at that OD). O1 is the OD value at which you want to start measuring the growth and O2 is the OD value of the high-density culture (OD = 0.8 -1). V1 is the volume of culture that has the OD=O1. V2 is the volume of the high-density culture that needs to be diluted to V1 in order to obtain a culture with OD=O1.
?. [Sample Preparation]
For the purposes of this experiment, we want to start measuring growth from a  culture of OD = 0.2. Let's say we measured the OD of the high-density culture to be 0.8. Thus, by the above formula; 0.2x100 = 0.8xV2V2 = 25 ml. Thus we need to dilute 25 ml of the high-density culture to   with the BG-11 media containing  NaCl.
100 mL
100 mL
?. [Making Measurements]
Once the  culture of OD=0.2 is obtained, we can start making measurements. Take  of this culture and record the absorbance value in the spectrophotometer.
100 mL
1 mL
?. [Making Measurements]
Take  of the culture and make absorbance measurements every 3 hours till an OD=1 is obtained.
1 mL
TITLE: RNAseq of primary human T cells overexpressing BATF3
AUTHORS: Andrea R Daniel

[DESCRIPTION]
This is a protocol describes methods for performing RNAseq using human CD8+ T cells overexpressing BATF3 or a GFP control.

[STEPS]
SECTION: Transfections for high-titer lentiviral production
1. Plate 1.2 x 106 or 7 x 106 HEK293T cells in a 6 well plate or 10 cm dish in the afternoon with 2 mL or 12 mL of complete opti-MEM (Opti-MEM‱ I Reduced Serum Medium supplemented with 1x  Glutamax, 5% FBS, 1 mM Sodium Pyruvate, and 1x MEM Non-Essential Amino Acids).
SECTION: Transfections for high-titer lentiviral production
2. The next morning, transfect HEK293T cells with 0.5 μg pMD2.G, 1.5 μg psPAX2, and 0.5 μg transgene for 6 well plates or 3.25 μg pMD2.G, 9.75 μg psPAX2, and 4.3 μg transgene for 10 cm dishes using Lipofectamine 3000.
SECTION: Transfections for high-titer lentiviral production
3. Exchanged media 6 hours after transfection and collect and pool lentiviral supernatant at 24 hours and 48 hours after transfection.
SECTION: Transduction of primary human CD8+ T cells
6. Centrifuged lentiviral supernatant at 600g for 10 min to remove cellular debris. 
SECTION: Transduction of primary human CD8+ T cells
7. Concentrate lentivirus to 50–100× the initial concentration using Lenti-X Concentrator (Takara Bio). 
SECTION: Transduction of primary human CD8+ T cells
8. Transduce T cells at 5–10% v/v of concentrated lentivirus at 24 h post-activation. For dual transduction experiments, T cells were serially transduced at 24 h and 48 h.
SECTION: Primary human CD8+ T cell cultures
4. Isolated CD8+ T cells from individual donors were obtianed directly from vials purchased from StemCell Technologies. 
SECTION: Primary human CD8+ T cell cultures
5. Culture T cells were in PRIME-XV T cell Expansion XSFM (FujiFilm) supplemented with 5% human platelet lysate (Compass Biomed), 100 U ml−1 penicillin and 100 μg ml−1 streptomycin. All media were supplemented with 100 U ml−1 human IL-2 (Peprotech). 
SECTION: RNAseq
9. T cells overexpressing either BATF3 or GFP were cultured for 10 days post transduction.
SECTION: RNAseq
10. RNA was isolated using Norgen’s Total RNA Purification Plus Kit and submitted to Azenta (formerly Genewiz) for standard RNA-seq with polyA selection.
SECTION: RNAseq
11. Reads were first trimmed using Trimmomatic72 v0.32 to remove adapters and then aligned to GRCh38 using STAR v2.4.1a aligner.
SECTION: RNAseq
12. Gene counts were obtained with featureCounts73 from the subread package (version 1.4.6-p4) using the comprehensive gene annotation in Gencode v22.
SECTION: RNAseq
13. Differential expression analysis was determined with DESeq2 (ref. 68) where gene counts are fitted into a negative binomial generalized linear model and a Wald test determines significant DEGs. 
SECTION: RNAseq
14. DESeq2 results of RNA-seq analyses with BATF3 OE and ZNF217 or GATA3 KO are presented in Supplementary Tables 4 and 7, respectively. 
SECTION: RNAseq
15. Upregulated and downregulated DEGs were input into EnrichR’s GO Biological Processes 2021 database71 for functional annotation.
TITLE: Ampure Bead Cleanup
AUTHORS: Alex Shaw, Manasi Majumdar, Catherine Troman, Javier Martin, Nick Grassly

[STEPS]
?. Prepare the AMPure XP beads for use; resuspend by vortexing.
?. Add required volume of resuspended AMPure XP beads to the reaction and mix by pipetting.
?. Incubate on a rotator for 5 minutes at room temperature.
?. Spin down the sample and pellet on a magnet. Keep the tube on the magnet, and pipette off the supernatant.
?. Keep on magnet, wash beads with 100 µL of freshly prepared 70% ethanol without disturbing the pellet.
?. Remove the 70% ethanol using a pipette and discard.
?. Spin down and place the tube back on the magnet.
?. Pipette off any residual 70% ethanol.
?. Briefly allow to dry.
?. Remove the tube from the magnetic rack and resuspend pellet in elutant.
?. Incubate for 2 minutes at RT.
?. Pellet beads on magnet until the eluate is clear and colourless.
?. Repeat steps 5 and 6.
?. Remove desired volume of eluate and store in a clean tube. Avoid disturbing the pelleted beads.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This is an example of a simple read mapping workflow. It is designed to be performed via the command line on an Ubuntu 16.06 OS. </p>
<p> </p>
<p>After completing this tutorial you should:</p>
<p>1) Have a practical understanding of how read mapping analyses are performed in the command line.</p>
<p>2) Understand the basics of how to process SAM and BAM files.</p>
<p>3) Be able to calculate depth of coverage of a contig/scaffold/chromosome in a query read dataset.</p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?.
TITLE: Plasma procurement and processing 
AUTHORS: hannah.anvari, asma.giornazi, shriya.shah, maryellen.pavone, francesca.e.duncan francesca.duncan

[DESCRIPTION]
Purpose: This protocol is intended for use in the collection and storage of human plasma in a research setting. The protocol details the collection, processing, and long-term storage for downstream analysis alongside other tissues or samples of interest (i.e. follicular fluid, ovary).

[GUIDELINES]
Researchers will adhere to all safety and training protocols required by the institution (Northwestern Medicine/Northwestern
University) including but not limited to:
Biosafety Certification
Bloodborne Pathogens Certification
Collaborative Institutional TrainingInitiative (CITI program)Certification


[STEPS]
1. Blood samples in BD Vacutainer™ Plastic Blood Collection Tubes with K2EDTA with Tube Stopper (Fisher Scientific, 23-021-013) are collected in pre-op from nurses. Samples are kept upright and placed on ice immediately following collection. Research coordinator will bring both specimens to the lab on ice (2-4 °C). Samples should be transported in blue, labeled biohazard cooler bag (Fig. 1A, B). 

  

2. Blood is centrifuged at 300g for 10min at room temperature (RT). Images of blood samples before and after centrifugation are seen below (Fig. 2A, B). Plasma (translucent top layer) is then aliquoted into 2 mL cryovial tubes (Fig. 2C) and stored at -80°C.
TITLE: 3D Reconstruction of Neurons in Vaa3D
AUTHORS: Allen Institute for Brain Science

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is used to generate accurate digital representations of neuron morphologies from a variety of brain regions and species. Each reconstruction captures the positions and thicknesses of the soma, dendrites and axon of a biocytin-filled cell within a slice of brain tissue. To generate the reconstruction we use an image stack containing ~200-700 serial 2D images that captures the full extent of the cell within the slice. We use the Vaa3D (Terafly) program with a Kazom’s Mozak user interface (Mozak for short) to visualize the 2D images in 3D. Once the stack is loaded in Mozak, our reconstruction is generated by placing nodes in 3D space. The placement of these nodes is dependent on the signal in the images. Our final output, an SWC text file (.swc format), contains many thousand rows. Each row contains a node ID, an x, y, z coordinate, radius value, neurite type, and parent node ID.  After tracing is complete, we preform post processing to provide radius values, check for errors and consistency then the SWCs are uploaded into our Laboratory Information Management System.</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: Blood collection for BALs
AUTHORS: Brent Boomhower, Olivier George

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol specifically outlines the blood collection procedure for gas chromatography BAL analysis by Dr. Amanda Robert's Lab at TSRI's Alcohol Research Center.</div></div>

[STEPS]
?. Submit a BAL request a day before collecting blood on https://www.oliviergeorge.com/balIACUC Protocol  : S19016Funding Source : ARCMethod : GCComments : State the time & date the samples will be delivered to TSRI
Ask mentor for directions to the specific drop-off site for blood samples.
?. Label 1.5 mL microcentrifuge tubes with corresponding animal IDs.
Animals must be bled within ~30 minutes of passive vapor shut off to prevent lowering of BALs before blood collection.
?. Open microcentrifuge tube and place in a tube rack that can hold all samples.
** The samples will be dropped off at TSRI in this tube rack. Label tube rack on tape with your name, "George," date, and number of samples. **
?. Pick up the animal and firmly hold its body under your armpit to restrain the animal, leaving its tail exposed.
Animal can also be restrained by wrapping the body in a towel or disposable underpad.
?. Place the distal tail on a flat surface and use an 18g needle to perpendicularly pierce ~1-2 cm from the tail tip all the way through the tail.
Use a new needle every few animals to maintain a clean puncture.
?. Immediately after puncturing, draw blood from the tail by firmly dragging fingers from the base of the tail to the end and repeat until ~100 ul of blood is collected.
?. Close the microcentrifuge tube and repeat steps 3-6 for all animals.
?. Centrifuge blood samples at 13,000 rpm and 22ºC for 10 minutes and place samples back into tube rack on wet ice.
?. Transport on wet ice to TSRI for gas chromatography analysis.
The door code at the sample drop-off area is 5142.
TITLE: Copy of     Direct ELISA for investigating the binding of  chemically-made Protein-LAG  to immunoglobulins.
AUTHORS: Angel  Justiz-Vaillant

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Protein LAG (SpLAG) is an immunoglobulin-binding protein that interacts with the Fc and Fab regions of many mammalian immunoglobulins. It is produced by a chemical coupling of individual proteins and them mixing it up to the appropriate protein ratio. SpLAG binds well to some avian immunoglobulin [1].</div><div class = "text-block">References</div><div class = "text-block">1.Vaillant AJ, McFarlane-Andersonv N, Wisdom B, Mohammed W, Vuma S, et al. (2013) Immunoglobulin-binding Bacterial Proteins (IBP) Conjugates and their Reactivity with Immunoglobulin in Enzyme-Linked Immunosorbent Assays (ELISA). J Anal Bioanal Tech 4: 175. doi:10.4172/2155-9872.1000175 </div></div>

[STEPS]
?. This ELISA is used to study the interaction of protein-LAG (SpLAG) with diverse immunoglobulins.
?. The 96 well microtitre plate is coated overnight at 4°C with 1 µg/µl per well of purified immunoglobulins or 50 µl of any animal sera in carbonate-bicarbonate buffer pH 9.6.
?. Then plate is treated with bovine serum albumin solution and washed 4X with PBS-Tween.
?. Then 50 µl of peroxidase-labeled-protein-LAG conjugate diluted 1:5000 in PBS-non-fat milk is added to each well and  incubated for 1.30h at RT. After that the plate is washed 4X with PBS-Tween.
?. Pipette 50 μl of 3,3',5,5' - tetramethylbenzidine (TMB; Sigma-Aldrich) to each well.
?. The reaction is stopped with 50 µl of 3M H2SO4 solution.
?. The plate is visually assessed for the development of colour and read in a microplate reader at 450 nm.
?. A cut-off point should be calculated as the mean of the optical density of negative controls x 2 SD.
TITLE: AccuBlue®Broad Range RNA Quantitation
AUTHORS: Ajit N Shah

[STEPS]
?. Warm all components to room temperature before use.RNA Broad Range Dyeis provided in DMSO, which may freeze during storage at 4°C.
?. Prepare 200 uL of working solution for each sample to be tested. Dilute the RNA Broad Range Dye in RNA Broad Range Buffer at a ratio of 1:200 in a plastic container and mix well by vortexing or shaking. For example, combine 10 uL of Dye with 2 mL Broad Range Buffer to prepare enough working solution for 10 tubes. Volumes can be scaled as required.
?. For each sample and standard, pipette 200 uL of the working solution into a clear 0.5 mL PCR tube.
?. Into one tube, pipet 10 uL of RNA Dilution Buffer (0 ng/uL).Into a second tube, pipet 10 uL of RNA Broad Range Standard (100 ng/uL).Pipette 10 uL of each RNA sample to be quantified in its own tube.  AB1Tubes2Standard 110 uL of RNA Dilution Buffer3Standard 210 uL of RNA Broad Range Standard 4Sample10ul of sample or Diluted sample
AB1Tubes2Standard 110 uL of RNA Dilution Buffer3Standard 210 uL of RNA Broad Range Standard 4Sample10ul of sample or Diluted sample
?. Incubate the tubes at room temperature for at least 2 minutes.
?. Turn on the Qubit® 3.0 instrument. On the home screen select RNA. Choose the Broad Range assay.Follow the prompts on the screen, and first read the tube containing RNADilution Buffer (ie, Standard 1) and then read the tube containing RNA Broad Range Standard (ie, Standard 2). The program will use these values to quantify your unknown samples.
?. The data can be recorded manually or exported as a csv file.
TITLE:       ELISA for quantification of IL-31 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>IL-31 is produced mainly by Th2 cells and dendritic cells. It is a proinflammatory cytokine and a chemotactic factor that direct polymorphonuclear cells, monocytes, and T cells to inflammatory lesions. IL-31 induces chemokines production and synthesis of IL-6, IL-16, and IL-32. </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-31 coating antibody is adsorbed onto the microwells by incubation overnight at 4°C with carbonate-bicarbonate buffer.
?. Add 50 µl of human serum. Human IL-31 present in the serum sample binds to antibodies adsorbed into 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-31 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-31 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) into each well.
?. Incubate the microwells in the dark for 15 min.
?. A colored product is formed in proportion to the quantity of human IL-31 present in the sample or standard.
?. The reaction is terminated by addition of 100 µl 3M H2SO4  and the absorbance is measured at 450 nm.
?. A standard curve is made from 7 human IL-31 standard dilutions and the human IL-31 sample concentration is determined.
?. For better results place the microplate on a microplate shaker in every incubation.
TITLE: Concentrate viruses from sewage using HA filters
AUTHORS: Adélaïde Roguet

[STEPS]
?. [Filtration of the sewage samples (in the biosafety cabinet)]
Take the sample out from the refrigerator.
?. [Filtration of the sewage samples (in the biosafety cabinet)]
Homogenize the sample thoroughly, avoiding foaming.
?. [Filtration of the sewage samples (in the biosafety cabinet)]
Use a pipette controller, transfer 25 mL of the sample into a 50-mL tube (where MgCl2 has been previously added). Repeat for each replicate. Save the rest of sample at 4ºC for archive or until decontamination and disposal.
[final concentration]
?. [Filtration of the sewage samples (in the biosafety cabinet)]
Spike in each replicate 5 µL of Bovine Coronavirus recovery control (~100,000 cp/uL), prepared as Bovine coronavirus solution prepared according to
?. [Filtration of the sewage samples (in the biosafety cabinet)]
Filter the samples in the 50-mL tubes:
?. [Filtration of the sewage samples (in the biosafety cabinet)]
Store filters at -80ºC for a minimum of 2h or overnight prior to the nucleic acid extraction. Archive filters at -80ºC.
?. [Filtration of the sewage samples (in the biosafety cabinet)]
Put the 50-mL tubes on ice until further processing.
?. [Filtration of the sewage samples (in the biosafety cabinet)]
Homogenize the sample in the 50-mL tube. Avoid as much as possible the liquid to touch the lid.
?. [Filtration of the sewage samples (in the biosafety cabinet)]
Filter the content in the 50-mL tube through a 0.80 µm 47mm cellulose ester filter at low vacuum pressure (~50 mmHg VAC).
?. [Filtration of the sewage samples (in the biosafety cabinet)]
As soon as the filtration is complete, transfer the filter to a 2-mL ZR BashingBead Lysis Tubes containing 650 µL of PM1 buffer solution (previously added).
It is important not to “over-dry” the filter on the filtration system.
Fold the filter as shown in Figure 1. Do not crease the filters with the forceps.
?. [Filtration of the sewage samples (in the biosafety cabinet)]
Place the HA filter in the filtration funnel. filter
Make sure the filter gets wet due to the residual water on the porous plate.
?. [Filtration of the sewage samples (in the biosafety cabinet)]
Rinse the clean filtration funnel with sterile deionized water.
Make sure some water is still on the porous plate.
?. [Filtration of the sewage samples (in the biosafety cabinet)]
Put the 2-mL tubes on ice until further processing.
?. [Filtration of the sewage samples (in the biosafety cabinet)]
Repeat Steps 6 for the other replicates.
?. [Nucleic acid extraction]
?. [Filtration of the sewage samples (in the biosafety cabinet)]
Store filters at -20ºC until all samples are processed
?. [Quantification]