eamag/protocols-with-steps huggingface dataset:
TITLE: Isolation of SARS-Cov2 RNA from Humans Without High Demand Reagents
AUTHORS: Joseph Patterson, Allyson Cole-Strauss, John Beck, Caryl Sortwell, Jack Lipton

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Viral RNA isolation kits used in PCR based tests for the novel coronavirus (SARS-Cov2) are in short supply. Our group sought to identify a method to isolate viral RNA without the use of a kit or other supplies in high demand by clinical labs. Using a TRIzol based RNA extraction, followed by a glycogen precipitation, we are able to isolate a sufficient quality and quantity of RNA for quantitative PCR, and/or droplet digital PCR. This method has been confirmed to work as well as a viral RNA isolation kit, shown to work with SARS-Cov2 RNA-spiked into human samples, and in detecting SARS-Cov2 in known positive cases of COVID-19. </div></div>

[STEPS]
?. Transfer the swab into a DNase/RNase free 1.5 mL microcentrifuge tube. •NOTE: The swabs we use can be pulled off their stick for easier processing.
?. Add 500 µL of TRIzol to the microcentrifuge tube.
?. Close tube and shake for 2 min. by hand.
?. Remove the swab and briefly centrifuge. •NOTE: The swab should be removed from the TRIzol, and squeezed against the side of the tube to retain as much sample as possible.
?. Add 100 uL of 100% chloroform to the phasemaker tube.
?. Shake the tube for 15 seconds by hand (DO NOT VORTEX).
?. Incubate for 10 min. at room temperature.
?. Centrifuge for 5 min. at 16,000xg at 4°C.
?. Transfer the aqueous phase (clear) to a new DNase/RNase free 1.5 mL microcentrifuge tube,  and dispose of the Phasemaker tube.
?. Add 250 µL of 100% isopropanol
?. Add 2 µL of GlycoBlue coprecipitant
?. Vortex for 30 s.
?. Centrifuge at 12,000 x g for 20 min. at 4 ºC.
?. Discard the supernatant, keep the pellet.
?. Add 80% ethanol to wash the pellet.
?. Immediately remove all of the ethanol. •NOTE: Once most of the ethanol is removed, the tubes can be briefly centrifuged and a pipet used to remove as much of the remaining ethanol as possible.
?. Allow pellet to dry for 3 min. at room temperature.
?. Resuspend pellet in nuclease free water.
?. Centrifuge Phasemaker tubes (Invitrogen A33248) for 30 s at 16,000xg
?. Transfer the entire sample to a Phasemaker tube and incubate at RT for 5 min
?. If Phasemaker tubes are not available, substitute the steps below for steps 6-11: 1)Add 100 uL of 100% chloroform.2)Vortex for 30 s.3)Incubate for 3 min. at room temperature.4)Centrifuge at 12,000 x g for 10 min. at 4 ºC.5)Transfer supernatant to a new DNase/RNase free 1.5 mL microcentrifuge tube. 6) Continue with protocol at step 7, adding 250 µL of 100% isopropanol to each tube.
?. Samples can be frozen at -80 ºC at this point if needed, then thawed for use when ready.
?. For both droplet digital PCR and qPCR protocols, 25µL nuclease free water is an appropriate starting volume, but can be adjusted if needed.
TITLE: Depression Detection with DM
AUTHORS: Umme Marzia  Haque

[DESCRIPTION]
The study has used data from YMM. The Yes/No variables that had a low correlation with target variable have been removed. To extract the most relevant features , the high correlated variables with the target variable , the Boruta method was used in conjunction with a Random Forest( RF) Classifier. To select suitable supervised learning models, the Tree-based Pipeline Optimization Tool To select suitable supervised learning models, the Tree-based Pipeline Optimization Tool (TPOTclassifier) has been employed. RF, XGBoost (XGB), Decision Tree (DT), and Gaussian Naive Bayes (GaussianNB) have been employed in the depression identification step.has been employed. RF, XGBoost (XGB), Decision Tree (DT), and Gaussian Naive Bayes (GaussianNB) were employed in the depression identification step.

[STEPS]
TITLE: Explant Surgery: Chronic recoverable Neuropixels in mice
AUTHORS: Emily A Aery Jones

[DESCRIPTION]
This protocol collection explains how to build a low-cost, lightweight system to implant Neuropixels 1.0 probes into mice, record during freely moving behavior, then recover the probe for future use. This protocol explains how to recover a previously implanted probe and clean for future use.

[STEPS]
SECTION: Prepare mouse
3. Set the nosecone to the position you recorded from the implant surgery. Attach mouse to headbar stereotactic adapters.
SECTION: Remove probe
4. Remove all the tape to expose the screws and probe.
SECTION: Remove probe
5. Cut the ground wire above the pin.
SECTION: Remove probe
6. Attach the Sensapex holder and set the stereotax to the angle used in the implant surgery. Tax to the dovetail cap. A fully open Sensapex holder can be taxed in from anterior or dorsal to the dovetail holder.
SECTION: Remove probe
7. Adjust the screws so the holder grips the dovetail cap.
SECTION: Remove probe
8. Unscrew the wings from the body. Screws can be saved for future surgeries.
SECTION: Remove probe
9. Slowly tax up to bring the probe & body piece away from the animal. Set back to 0 degrees and adjust the arm so the probe is away from the surgical field.
SECTION: Remove probe
10. Lower the probe into tergazyme and let sit for 30 minutes, then rinse with dH2O.
SECTION: Perfuse
11. Remove the mouse from the stereotax and prepare for perfusion.
  

SECTION: Prepare mouse
1. Set O2 flow rate to 1-1.5L/min and isoflurane to 3%. Place mouse in anesthetic chamber.
 

SECTION: Prepare mouse
2. When breathing has slowed to 1Hz, move animal to the toothbar. Switch isoflurane from chamber to nosecone. Wait until unresponsive to pedal reflex test, then lower to 1.5% isoflurane.
 



SECTION: Re-use probe
12. Assemble following the Assembly protocol, skipping the steps that modify the probe (sharpening, soldering wire to the PCB, attaching the body piece). Extend the ground wire back to full length and attach a new pin.
TITLE: TSS competent cells and transformation 
AUTHORS: David Ruano Gallego

[DESCRIPTION]
This protocol allows the efficient transformation of plasmid into E. coli and related strains.

[BEFORE_START]
Prepare solution TSS and KCM.

When prepared, TSS has a cloudy appearance that disappears once autoclaved. Also, over time, TSS can seem to be contaminated without being so, since the polyethylene glycol tends to precipitate.

[STEPS]
SECTION: Preparing competent cells
1. Grow o/n a 5 mL culture of the strain to be transformed.
5 mL 37 °C   
SECTION: Preparing competent cells
2. Dilute 1:100 of the preinoculum in 50 mL prewarmed LB (OD600 = 0,05).
50 mL 37 °C 
SECTION: Preparing competent cells
3. Grow at 37ºC with agitation (170 rpm) until it reaches OD600 = 0,5.
 
SECTION: Preparing competent cells
4. Centrifuge the culture at 4000 g and 4ºC for 10 min.
4000 x g, 10 min, 4 °C 
SECTION: Transformation protocol
15. Recovery: add 250 µl of LB and incubate at 37ºC for 1 h.
37 °C 60 min 
SECTION: Transformation protocol
16. Plate 100 µl of the solution on LB agar plates supplemented with the indicated antibiotic and incubate o/n at 37ºC.
37 °C   
SECTION: Preparing competent cells
5. Discard supernatant and wash with 1 mL of chilled TSS.
1 mL on ice 
SECTION: Preparing competent cells
8. Incubate 10 min on ice.
on ice 10 min 
SECTION: Preparing competent cells
7. Discard supernatant and resuspend in 1 mL of TSS.
1 mL on ice 
SECTION: Transformation protocol
12. Incubate 30 min on ice.
on ice30 min  
SECTION: Transformation protocol
13. Thermal shock: incubate at 42ºC for 90 s.
42 °C 90 s 
SECTION: Preparing competent cells
9. Store 30 µL aliquots at -80ºC.
30 µL -80 °C 
SECTION: Preparing competent cells
6. Centrifuge at 4000 g and 4ºC for 3 min.
4000 x g, 3 min, 4 °C 
SECTION: Transformation protocol
10. Mix 5 μL of 5x KCM solution with DNA (around 1-5 μL) and H2O to a total of a 25 μL mixture.25 µL 
SECTION: Transformation protocol
11. Mix the mixture with 25 μL of competent cells.
25 µL 
SECTION: Transformation protocol
14. Incubate 2 min on ice.
on ice 2 min
TITLE: Differential transcript usage analysis
AUTHORS: Peter Kilfeather

[DESCRIPTION]
Differential transcript usage analysis from Kilfeather, Khoo et al., 2024

[STEPS]
SECTION: Protocol
1. Differential transcript usage was performed using DRIMSeq (v1.24.0). To consider only genes containing more than 1 isoform, with a minimum level of expression, the following parameters were used: min_samps_gene_expr = n * 0.75, min_gene_expr = 10, min_samps_feature_expr = 10, min_feature_expr = 10, min_samps_feature_prop = 10, min_feature_prop = 0.1.
TITLE: 0.9% Saline Solution
AUTHORS: Allen Institute for Brain Science

[DESCRIPTION]
This protocol is used to prepare 0.9% Saline Solution. 0.9% Sterile Saline solution is used as an initial rinse for the brains of mice prior to perfusion. 

Note: 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.

[STEPS]
TITLE: GUARDS Statistical Analysis Plan Version 1.1 
AUTHORS: Professor Catherine Williamson, Dr Jose Blanco Carnero, Dr Catalina de Paco Matallana, Dr Alice Mitchell, Dr Caroline Ovadia, Professor David Wright, Monika Lewanczyk

[DESCRIPTION]
GUARDS is a double-blind randomised placebo-controlled single site trial. The participating centre in Spain will be Clinic University Hospital “Virgen de la Arrixaca” in Murcia. Women with an abnormal oral glucose tolerance test (OGTT) screen will be offered participation. After written informed consent, eligible women will be randomly assigned to UDCA or placebo at 24+0-30+6 weeks’ gestation until birth. All participants will be offered standard antenatal care, aiming for the GEDE guideline targets of blood glucose concentration between 3.9-7.8mmol/L (70 - 140 mg/dL). We will assess clinical efficacy, mechanisms of action and acceptability of UCDA in study participants. Clinical efficacy will be measured by maternal-infant glucose control using conventional capillary and novel continuous glucose measures and standard clinical measures for serum lipids (total, HDL- and LDL-cholesterol, triglycerides and total free fatty acids). Acceptability will be measured by patient compliance (pill counts) and confirmed by serum measurements of individual bile acids (including UDCA). 

Serum samples will be taken for UPLC-MS/MS measurement of bile acids (including UDCA), so it will be clear if women have taken (and absorbed) the drug. Obstetric and neonatal outcomes will be measured. This Statistical Analysis Plan sets out how we will analyse the trial.

[STEPS]
TITLE: Bodo saltans transfection with plasmid for tagging EF1alpha gene__IG BsTub
AUTHORS: Fatma Gomaa, Roberto Docampo, Zhuhong Li, Peter Girguis, Virginia Edgcomb

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Stable transfection of </span><span style = "font-style:italic;">B. saltans</span><span> has been achieved using plasmid  for EF 1 alpha C terminal tagging.</span></div></div>

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
For use in <a href="https://www.protocols.io/view/Polyacrylamide-Gel-System-For-Electrophoresis-Of-P-ep7bdrn" target="_blank">Polyacrylamide Gel System For Electrophoresis Of Proteins</a>.

[STEPS]
?. 
?. 
?. 
?.
TITLE: Detection of cytosolic and mitochondrial ROS production using FlexStation 3
AUTHORS: Nawel Zaibi; Pengyun Li; Shang-Zhong Xu

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is for the detection of cytosolic and mitochondrial ROS using FlexStation 3</div></div>

[STEPS]
?.
TITLE: A novel laboratory method to simulate climatic stress with successful application to experiments with medically relevant ticks
AUTHORS: Sang Hyo Kim, Caleb  Nielebeck, Lauren Dedmon, Mark Pangilinan, Jahred Quan, William Ota, Javier  D. Monzón

[DESCRIPTION]
This protocol details a novel method to isolate individual ticks and manipulate their environment. We successfully used this method to investigate how humidity affects survival and host-seeking (questing) behavior of three species of ticks: the lone star tick (Amblyomma americanum), American dog tick (Dermacentor variabilis), and black-legged tick (Ixodes scapularis). We placed 72 adult females of each species into individual plastic tubes and separated them into three experimental relative humidity (RH) treatments representing distinct climates: 32% RH, 58% RH, and 84% RH. For 30 days we assessed the survival and questing behavior of each tick.

[BEFORE_START]
We acquired adult female ticks from the Oklahoma State University Tick Rearing Facility and experimented with one temperature range and three relative humidities. This protocol can be modified for different species, life stages, temperatures, humidities, and other small organisms. 

[GUIDELINES]
Questing qualifications: 
The individual is not walking but it is still with its front legs extended       

Death qualifications: 
If any tick appears dead, lightly blow on it since ticks respond to carbon dioxide exhaled by potential hosts
If the tick does not move at all in 2 minutes, it should be counted as dead and placed in 70% ethanol

[STEPS]
SECTION: Set up
1. Place a single tick with one wooden skewer in each tube and seal with a cap, labelling each tube with an individual identifier  
  
SECTION: Set up
2. Place six tubes in each airtight container along with a humidity pack, labelling each container
  

SECTION: Set up
3. Program the climate chamber
 

SECTION: Set up
3.1. To cycle between 20 °C to 30 °C, the temperature increments should be as follows: 
3:00  - 25 °C 
6:00  - 27.5 °C 
9:00  - 30 °C 
12:00  - 27.5 °C 
15:00  - 25 °C 
18:00  - 22.5 °C 
21:00  - 20 °C 
24:00  - 22.5 °C 


SECTION: Set up
3.2. To create a 12:12 light:dark photoperiod, lighting increments should be as follows:
9:00 - Lights on
21:00 - Lights off
SECTION: Data collection
4. Place all of the airtight containers, filled with ticks and humidity packs, into the climate chamber and start the program
SECTION: Data collection
5. Each day thereafter, during the 9:00 to 12:00 or 30 °C increment, assess each tick for survivorship and questing behavior (see guidelines for qualifications of survivorship and questing)
 

 


SECTION: Set up
2.1. Confirm the humidity in one container of each RH level with the data logger 

SECTION: Data collection
6. Repeat steps 4 and 5 for 30 days or until all ticks have died
SECTION: Data collection
5.1. Periodically move the data logger to a new bin to confirm that no unexpected changes to the climate inside the containers has occurred
TITLE:    ELISA for quantification of IL-8 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>Monocytes and fibroblasts make IL-8. Its principal targets are neutrophils, basophils, mast cells, macrophages, and keratinocytes. It causes neutrophil chemotaxis, angiogenesis, superoxide release, and granule release.</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-8 coating antibody is adsorbed onto microwells by incubation overnight at 4°C.
?. Add 50 µl  of human serum. Human IL-8 present in the serum sample binds to antibodies adsorbed to the microwells.
?. The microplate is blocked with 3% non-fat milk-PBS buffer and later wash to remove unbound proteins.
?. Fifty (50) µl of biotin-conjugated anti-human IL-8 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-8 antibody. The optimal dilution of this conjugate must be investigated.
?. The plate is washed following incubation to remove the unbound Streptavidin-HRP.
?. Add 100 µl of 3,3',5,5'- tetramethylbenzidine (TMB; Sigma-Aldrich) to each well.
?. Incubate the microwells in the dark for 15 min.
?. A colored product is formed in proportion to the quantity of human IL-8 present in the sample or standard.
?. The reaction is terminated by addition of 100 µl 3M H2SO4  and absorbance is measured at 450 nm.
?. A standard curve is made from 7 human IL-8 standard dilutions and the human IL-8 sample concentration determined.
?. For better results place the microplate on a plate shaker in every incubation.
TITLE: Brain Histology - tissue sectioning and staining
AUTHORS: Yiqin Shen

[DESCRIPTION]
.

[STEPS]
SECTION: Sectioning
1. Turn on and set the water floating device to 37 °C .
Prepare a bucket of ice. Place the samples on ice before sectioning.
Check the blades by doing a few test sections. Change if they are not sharp. 
SECTION: Staining- Day 1
5. Place the slides onto the slide holder, and dip in xylene for 30 min.
SECTION: Staining- Day 1
6. Wash the slides with alcohol. The washes are at different concentrations from 100% to 50%, 6 min each.
Rinse with DD H2O
SECTION: Staining- Day 1
7. Remove the slides from holder, trace the outside with hydrophobic pen. 
SECTION: Staining- Day 1
8. Add blocking buffer (Bloxall) to the slides for  30 min. 
Rinse with PBS 2X 3min

SECTION: Staining- Day 1
9. Place the slides onto the holder. It should be cooked with pressure cooker in citrate buffer for 25 min.
SECTION: Staining- Day 1
10. Remove the slides from the holder and place them down.
Add 2.5% horse serum to the slides for 25 min.
Wash with PBS 2X 3 min.
SECTION: Staining- Day 1
11. Dilute primary antibody in blocking buffer, add to the slides. 
Incubate in the fridge 25 min 4C
SECTION: Staining- Day 2
12. Rinse the slides with PBS.
Dip in TBS-T for 10 min for three times. 
Rinse with PBS.
SECTION: Staining- Day 2
13. Add blocking buffer to the slides for  60 min

SECTION: Staining- Day 2
14. Add secondary antibody to the slides, at Room temperature 60 min.

SECTION: Staining- Day 2
15. Rinse the slides with PBS.
Dip in TBS-T for 10 min for three times. 
Rinse with PBS.
SECTION: Staining- Day 2
16. Develop color with chromogen, rinse in DD H2O to stop reaction
SECTION: Staining- Day 2
17. Put the slides in Hematoxylin for 30s
Rinse 2X DD H2O, and allow the slides to air-dry.
SECTION: Staining- Day 2
18. Put the slides in Xylene for 1 min two times.

SECTION: Staining- Day 2
19. Coverslip + mounting media
SECTION: Sectioning
2. Place a block of sample on the holder. 
Adjust the machine so the block is near the blade but not cut. Rotate the handle to bring near the block.
SECTION: Sectioning
3. Start sectioning. Avoid ruptures and curling.
 

SECTION: Sectioning
4. Place the slice on to water with brush, and gently catch them with glass slides. Label each slide well.
Allow the slides to air dry.
TITLE: SCC compressive strength test
AUTHORS: Guoqiang Du,  Liangtao Bu,  Qi Hou,  Jing Zhou

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This paper mainly introduces the test process of rebound method and ultrasonic method for high strength self-consolidating concrete with strength grade of C50, C60, C70, C80, C90 and C100, and carries out cube compressive strength test on the test cubes under the same conditions. The entire test process, equipment and materials are introduced.</div></div>

[STEPS]
TITLE: RNAseq DEG analysis protocol for non-human samples using ONT reads
AUTHORS: Avni Arora

[DESCRIPTION]
Data analysis of short-read sequencing from ONT flow cells can be done in a multitude of ways depending on the type of analysis that needs to be done and the sequencer utilized for assembly. Packages like NanoPack are available for use via Python however, there are fewer short-read sequencing packages available in RStudio. This protocol demonstrates how to analyze short-read transcriptome data in Rstudio and Python without the use of cloud-based software like EPI2ME. 





[BEFORE_START]
You may need to activate a conda environment and/or install python as well as RStudio in order to run these commands. 


[GUIDELINES]
This protocol was developed for the purposes of BIT 495: Special Topics in Biotechnology: Portable Genome Sequencing at North Carolina State University. 


[STEPS]
SECTION: Data clean up and alignment
1. Obtain FASTQ files from flow cell (GridION, MinION, PromethION) 
SECTION: Data clean up and alignment
2. Upload FASTQ files to machine 
SECTION: Trimming
3. Utilize the Prowler options to build a script to trim your reads based on the adapter lengths produced by the Fasta file. 
 

 

SECTION: Trimming
3.1. 
 

SECTION: Alignment and Quantification
4. 
 

SECTION: Alignment and Quantification
5. Creating reference index

 

 

SECTION: Alignment and Quantification
5.1. 
 

SECTION: Alignment and Quantification
6. Aligning to reference index 

 

 

SECTION: Alignment and Quantification
7. 

 

 

SECTION: Alignment and Quantification
9. Quantification using Salmon alignment based mapping 
 

 

SECTION: Alignment and Quantification
8. Quantification prior to summarization to the counts level can be done via Salmon using the alignment-based method. For ONT reads, the option –noLengthCorrection MUST be used. 
Download the transcriptome file for your reference organism


SECTION: Alignment and Quantification
10. Import non-normalized, raw count files to local machine and upload to Rstudio 


SECTION: Differential expression analysis : RStudio
11. 
 

SECTION: Differential expression analysis : RStudio
12. Summarization to counts level using Tximport 


 


SECTION: Differential expression analysis : RStudio
13. Once tximport is installed generate a sample sheet to organize your quant.sf files 


SECTION: Differential expression analysis : RStudio
14. Utilize the tximport vignette to generate script
tximport: Import and summarize transcript-level estimates for transcript- and gene-level analysis

SECTION: Differential expression analysis : RStudio
15. Differential expression analysis via DEseq2 

 

16. Utilize the DESeq2 vignette to generate script for differential expression analysis!

DESeq2: Differential gene expression analysis based on the negative binomial distribution
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocole provide guidance to detect<em> Mycobacterium ulcerans</em> in environmental samples. The protocole share the DNA extraction process using the FastDNA kit for soil samples and the amplification of gene based on a validated protocole published by Fyfe which is the most protocole use to date for such analyis.</p>

[BEFORE_START]
<p>All samples should be stored in refrigerator in +2ºC to +8ºC. The day of the analysis, samples must be left at the laboratory temperature (22ºC to 25ºC) before used. </p>

[GUIDELINES]
<p>DNA extraction should be performed before the PCR analysis.</p>
<p>The extract process should be adapted to the samples type and strictely adhere to the manufacturer recommendations.</p>

[STEPS]