eamag/protocols-with-steps huggingface dataset:
TITLE: Production of Recombinant EnvA Rabies Virus
AUTHORS: Allen Institute for Brain Science

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is used for the large-scale production of EnvA-pseudotyped recombinant rabies virus.</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: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol describes the library preparation for Nanopore sequencing according to the SQK-MAP005 protocol. </p>
<p>It accompanies the <em>GigaScience</em> publication:</p>
<p> </p>
<p>Benjamin Istace, et al. (2017) De novo assembly and population genomic survey of natural yeast isolates with the Oxford Nanopore MinION sequencer. <em>GigaScience...</em></p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: Reducing Power
AUTHORS: Jing Xu

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>The reducing power of samples were assayed using the Guo et al.1 ml of the extractwas added to 2.5 ml of phosphate buffer (0.2 M, pH 6.6) and 2.5 ml of 1% potassium ferricyanide.After 20 min, 2.5 ml of 10% trichloroacetic acid (TCA)was added, followed by centrifugation at 3000 rpm for 10 min. The upper layer (2.5 ml) was mixed with 2.5 ml of distilled water and 0.5 ml of 0.1% ferric chloride, after 10 min, the absorbance was measured at 700 nm. EC</span><span style = "vertical-align:sub;">50</span><span> was calculated from the graph of an inhibition percentageagainst extract concentration. </span></div></div>

[STEPS]
TITLE: Value of Information in Telehealth for Chronic Heart Failure
AUTHORS: Andrija S. Grustam, Nasuh Buyukkaramikli, Ron Koymans, Hubertus J.M. Vrijhoef, Johan L. Severens

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Figure 1 - Single loop Monte Carlo scheme for computing overall individual Expected Value of Perfect Information (EVPI). </span></div><div class = "text-block">and</div><div class = "text-block"><span style = "font-weight:bold;">Figure 2 - Sampling algorithm for the calculation of Expected Value of Partially Perfect Information (EVPPI) with double loop. </span></div></div>

[STEPS]
?.
TITLE: ASTROCYTE PRODUCTION (Support Protocol 7.1)
AUTHORS: Michael S. Fernandopulle, Ryan Prestil, Christopher Grunseich, Chao Wang, Li Gan, Michael E. Ward

[STEPS]
?. Use 3 P0 or 1 P1 rat pup per uncoated T75 flask. Meninges should be completely removed from brains, and astrocytes isolated per standard mechanical and/or enzymatic dissociation protocols under sterile conditions. Expand astrocytes for 1 week or until confluent in DMEM containing 10 % FBS by volume (astrocyte medium).
?. Shake at 300 rpm at  to remove microglia, any surviving neurons, and other contaminating cell types.
37 °C
?. When the flask nears confluency, wash with PBS and incubate with trypsin for .
?. Centrifuge  at , .
0 Room temperature
?. Seed cells from each T75 into three new T75 flasks and grow until nearly confluent.
Astrocytes may be passaged in culture up to two times. Passaging more than two times reduces neutrophic and synaptogenic support qualities.
?. Alternatively, astrocytes can be frozen in liquid nitrogen (see Basic Protocol 1).
?. To add astrocytes to neural cultures, repeat steps 3 to 6 or thaw from frozen stock, and seed cells onto a transwell.
Over-adding astrocytes is better than under-adding; if insufficient number of astrocytes are plated, neurons will likely be less healthy. The induction can largely be rescued by adding additional astrocytes the following day if it appears that the neurons are not responding well.
?. Aspirate supernatant.
?. Resuspend in DMEM containing 10 % FBS.
TITLE: ROTAROD PROTOCOL
AUTHORS: Michael Lee

[DESCRIPTION]
This protocol details the rotarod test.

[GUIDELINES]
Overview
Run groups of up to 5 animals, in a maximum of 4 groups in a single run of 4 trials (i.e. must have ~15-20 min between trials).
Run 4 trials for every animal each day for 4 days.
Detailed records should be kept on every experiment, noting date, timing, subject test order, procedural details followed and any relevant observations pertaining to the assay (e.g. specific behavior or procedural errors).

[STEPS]
SECTION: Set-up
1. Plug in Rotarod, turn on power switch in back.
SECTION: Set-up
2. Press mode (F1).
SECTION: Set-up
3. Press Acceleration (F2).
SECTION: Set-up
4. Set Ramp Duration to 5 min.

SECTION: Set-up
5. Press ESC.
SECTION: Set-up
6. Reverse → No.
SECTION: Set-up
7. Press Alt → Speed and set to a max. of 50 (default is 80).
SECTION: Set-up
8. Press Forward, and set to a min. speed of 5.
SECTION: Set-up
9. Press Reset for rotation (without acceleration).
SECTION: Set-up
10. Place animals of rotating rod by holding their tail (use a gentle sweeping motion to place animal).
SECTION: Set-up
11. Make sure paddles are up and the time is set at 0 for all animals.
SECTION: Set-up
12. Press Start.
 

SECTION: Clean-up
13. 
 
Remove the paddles and clean any animal droppings and urine.
SECTION: Clean-up
14. Place paddles back on Rotarod.
SECTION: Clean-up
15. Press Reset.
SECTION: Clean-up
16. Place next group of animals on rod.
SECTION: Clean-up
17. Flip paddles upward.
SECTION: Clean-up
18. Press Start.
SECTION: Data
19. For each day, calculate the average time and average number of rotations over the 4 trials for each animal.
TITLE: Glyoxal fixation of mammalian cells for immunofluorescence 
AUTHORS: Karla LH Feijs

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This is our standard protocol for glyoxal fixation of mammalian cells grown on glass cover slips for immunofluorescence microscopy.</div></div>

[STEPS]
?. [Fix the cells]
Wash the cells with warm DMEM without FCS.
?. [Fix the cells]
Add 300µl glyoxal solution per well (12-well plate) and incubate on ice for 30 minutes , then at room temperature for 20 minutes  .
on ice
0 Room temperature
?. [Fix the cells]
Wash 2 times with PBS
?. [Fix the cells]
Add 300µl quenching solution for 20 minutes
0 Room temperature
?. [Fix the cells]
Wash once with PBSAt this stage, fixed cells can be stored in the fridge for at least a week.
?. [Staining the cells]
Add 500µl blocking solution and incubate at room temperature for 30 minutes In meantime, prepare the primary antibody. The dilution is dependent on the antibody, but can range from undiluted for non-purified hybridoma supernatants to 1:1000 for affinity purified antibodies. The antibodies are diluted in antibody buffer.
0 Room temperature
?. [Staining the cells]
There are two ways to incubate the antibodies:1. Prepare enough so that the slide can be covered in the well with the antibody solution, ±250-300µl.2. To save antibody, prepare only 50µl and pipette this onto parafilm. Place the coverslip on top of this drop with the cells facing the drop and incubate in a humid chamber.Incubate at least 45 minutes at room temperature or alternatively overnight at 4ºC
0 Room temperature
?. [Staining the cells]
Place the coverslips back in the 12-well plate with the cells facing upwards.Wash 3 times with PBS.
?. [Staining the cells]
Dilute the secondary antibody in antibody buffer, generally in a range of 1:1000-1:2000 for most antibodies. Add 250-300µl per well and incubate for at least 45 minutes at room temperature or alternatively overnight at 4ºC  .IMPORTANT: this incubation step should be in the dark, as the conjugated secondary antibodies are light-sensitive.
0 Room temperature
?. [Staining the cells]
Wash 2 times in PBS, followed by 2 times in ddH2O.
?. [Staining the cells]
Place a drop of mountant containing DAPI on a microscope slide and place the coverslip on this drop with the cells facing the drop.IMPORTANT: warm up the mountant to 37ºC to avoid unequal DAPI-staining. Tip the coverslip sideways on a tissue to remove excess water before mounting.
?. [Staining the cells]
Dry the slides overnight in a dark place at room temperature. Store at 4ºC afterwards until imaging.
TITLE: Gait and rehabilitation in lower limb amputees: a narrative review
AUTHORS: Irene Aprile, Marco Gallotti, Marco Germanotta, Pasquale Alessio  Sauchelli

[DESCRIPTION]
Introduction:Even if gait analysis is a validated outcome measure to assess the effects of a rehabilitation program on gait performance, few articles have been produced about the evaluation of a rehabilitation program in lower limb amputees using the gait analysis as a measure of outcome.
Inclusion criteria:The inclusion criteria of the following narrative review is to investigate all the studies, without year limitations but only in English, whose population was lower limb adult amputees that underwent a rehabilitation process evaluated through the use of the gait analysis as a measure of outcome.
Methods:Three databases will be investigated: Scopus, Pubmed and Embase, without year limitations but only in English. Study selection will be done following the inclusion and exclusion criteria, data extraction will follow the review’s objectives. Agree II scale will be used to assess the quality of guidelines, Amstar 2 scale will be used to evaluate systematic review, Pedro scale will be used to assess quality of randomized controlled trial, Stard-2015 scale will be used to assess observational studies. Two reviewers will screen the articles in blind and a third reviewer will solve the conflicts raised.
Objective:The aim of this narrative review will be the investigation of the extent, type and results of evidence in relation to gait and rehabilitation in lower limb amputees.  

[GUIDELINES]
Sagawa, Y., Turcot, K., Armand, S., Thevenon, A., Vuillerme, N., & Watelain, E. (2011). Biomechanics and physiological parameters during gait in lower-limb amputees: A systematic review. InGait and Posture(Vol. 33, Issue 4). https://doi.org/10.1016/j.gaitpost.2011.02.003

Prinsen, E. C., Nederhand, M. J., & Rietman, J. S. (2011). Adaptation strategies of the lower extremities of patients with a transtibial or transfemoral amputation during level walking: A systematic review. InArchives of Physical Medicine and Rehabilitation(Vol. 92, Issue 8). https://doi.org/10.1016/j.apmr.2011.01.017

[STEPS]
TITLE: Phenol-Chloroform Extraction for dsRNA Purification
AUTHORS: Cera Fisher

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This is the final step in preparing injectable constructs for RNA interference. This protocol starts with a 20 uL T7-polymerase transcription reaction. We use New England Biolab's HiScribe kit. Our template cDNA has been prepared with T7 promoter regions at each 5' end, enabling RNA transcription in both directions. After dsRNA has been extracted, pelleted, and resuspended in pure nuclease-free water, we will melt the strands apart and allow them to reanneal slowly to ensure that we have plenty of properly double-stranded RNA. </div><div class = "text-block">The protocol is based on the manufacturer's manual for the HiScribe kit, modified with information from the MegaScript T7 RNA transcription kit manual (Ambion). </div></div>

[STEPS]
?. [Adjust salt conditions]
Add 166.6 uL of nuclease free water to each PCR tube. Move 187 uL of liquid to new, labeled 1.5 mL microcentrifuge tube.
?. [Extract RNA]
Add 200 uL of acid phenol/chloroform (50:50) to each tube. Shake vigorously for 15 seconds.
?. [Extract RNA]
Centrifuge at 4C for 5 minutes at 12,000 * g or greater
?. [Extract RNA]
Move aqueous phase (top) to new tube (about 200 uL).
?. [Extract RNA]
Add an equal volume chloroform to the aqueous phase. Shake vigorously for 15 seconds.
?. [Extract RNA]
Centrifuge at 4C for 5 minutes at 12,000 * g or greater
?. [Extract RNA]
Move aqueous phase (top) to new tube
?. [Precipitate RNA]
Add an equal volume of isopropanol.
?. [Precipitate RNA]
Incubate at room temperature for 10 minutes
?. [Precipitate RNA]
To pellet: Spin at 4C for 20 minutes at 12,000 * g or greater
?. [Precipitate RNA]
Remove isopropanol and Wash pellet with 75% ethanol in nuclease free water
?. [Precipitate RNA]
Spin at 4C for 5 minutes at 7,500 * g
?. [Precipitate RNA]
Remove all ethanol from pellet and let air dry (for about 15 minutes)
?. [Melt and anneal dsRNA]
Resuspend dried pellet in 50 uL of nuclease free water.
?. [Melt and anneal dsRNA]
Heat resuspended dsRNA to between 65 and 80 degrees C for 5 minutes
?. [Melt and anneal dsRNA]
Allow to cool at room temperature for 10 minutes
?. Place on ice.
?. [Adjust salt conditions]
Add 13.33 uL of ammonium acetate to each tube.
TITLE: The Cluster Feature of Coalmine Disasters and Earthquakes in China
AUTHORS: Chen Bo

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>      Recently, in China, nearly half of coalmine disasters are found to present cluster feature or to be accompanied with earthquakes (<5) nearby, in which all the disaster types are involved. Of all the causative factors, stress disturbance, the only one that can cause all kinds of coalmine disasters at one time, seems to exist among mining areas and to be responsible for the cluster feature. In further research, more coalmine disasters are found accompanied with micro-earthquakes, which may become the vital geophysical evidence of the tectonic stress fluctuation around mining areas. This paper lists and analyzes all the possible causative factors to demonstrate the authenticity and reliability of the new phenomena and ultimately, all the other known causative factors have been speculated and excluded. A quantitative research has been made on the cluster degree, by which the space-time distribution curves are achieved. The readers can query the ratio of disasters involved in cluster series and the number of coalmine disasters accompanied with earthquakes. For example, under the threshold of </span><span style = "font-weight:bold;">10</span><span>0 kilometers, 47 % of disasters are involved in cluster series and 372 coalmine disasters are accompanied with earthquakes. The readers can also query the detailed distribution of the durations. Potentially, the duration of cluster series reflects that of stress disturbance. The majority cluster series lasted for 1~2 days, which are more likely related to local stress disturbance. While the minority lasting longer than 4 days correspond well with fatal earthquakes, which are speculated to be related to regional stress disturbance. In addition, all the cluster series under every threshold have been picked out successfully and a full </span><a href="http://pan.baidu.com/s/1sjnCEh3" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">open access</span></a><span> is provided. The cluster series possess multiple properties, such as the area, the related disasters, etc., which compared with the energy and the magnitude of earthquakes, good correspondences are acquired. It indicates that the cluster series of coalmine disasters and earthquakes are linked with fatal earthquakes and may serve as footprints of stress disturbance. The paper is not anxious to set up or to prove a theory, but to makes the most common speculations on a given geological model, in which five affecting cases are analyzed. To earthquake research and disaster prevention, widely scientific significance is suggested.</span></div></div>

[STEPS]
TITLE: sample_prep_serum.nan
AUTHORS: NAN KB, Mario Uchimiya, John Glushka, Christopher Esselman, Leandro I Ponce, Laura Morris, Arthur Edison

[DESCRIPTION]
This is a modified protocol for a protein precipitation method for plasma/serum samples. This protocol was originally proposed by:
 
See also:
 

 


[STEPS]
17. 
1.2. Add 600 µL of 100% cold methanol to 300 µL of samples on ice
Use 1.5-mL Eppendorf tubes
Keep methanol cold on ice

1.3. Vortex the samples for 10 s

1.4. Incubate the samples at -20 °C for 20 min 

1.5. Centrifuge the samples at 4 °C at   for 30 min 

1.6. Transfer the supernatants to new 1.5-mL Eppendorf tubes 
1.7. Dry the samples in a speed-vac concentrator  

 

2. Day-2/2
2.1. Thaw the samples on ice or in 4 °C
2.2. Add 600 µL of the phosphate buffer to each sample

2.3. Vortex the samples at 4 °C for 10 min
2.4. Centrifuge the samples at 4 °C for 10 s 
1.1. Thaw samples on ice or at 4 °C 

2.5. Transfer 580 µL to 5-mm NMR tubes

 

1. Day-1/2
TITLE: U Mass - Urea/BUN
AUTHORS: Jason Kim

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary: </span></div><div class = "text-block">This experiment involves a spectrophotometric measurement using Roche Cobas Clinical Chemistry Analyzer.  Serum Urea/BUN levels are affected by alterations in systemic protein and nitrogen metabolism.  Serum Urea/BUN levels are altered in kidney failure and renal complications of diabetes.</div></div>

[STEPS]
?. Perform daily quality control assessment of instrumentation before analysis.
?. Load each sample into a specialized micro-sample cup for the clinical chemistry analyzer.
?. Select Urea/BUN test on display and run the analysis.
?. Collect and analyze the data.
TITLE: Y Maze Forced Alternation
AUTHORS: Haley   Geertsma

[DESCRIPTION]
This protocol is used to test mice in the Y Maze Forced Alternation behavioural test.

[STEPS]
1. Habituate mice in a separate room for 60 minutes prior to testing.
2. Confirm testing room lighting is set to 60lux and EthoVision is set up properly.
3. Place mice in Arm 1 (with either Arm 2 or 3 blocked, alternate between mice) and record their movement for 5 minutes.
4. After testing, place the mouse back into the home cage and clean the Y maze. Leave the mouse in its home cage for 30 minutes.
5. Remove the blockage so all 3 arms are open and place the mouse back into Arm 1. Record their movement for 5 minutes.
6. Once testing is complete, return mouse to its home cage and clean the Y maze.
TITLE: Molecular Phenotype Distribution of Single Rat ICN Neurons - Heart B
AUTHORS: Shaina Robbins, Alison Moss, Sean Nieves, Sirisha Achanta

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This collection of protocols were used to obtain the current data for the Blackfynn Dataset Molecular Phenotype Distribution of Single Rate ICN Neurons, which we refer to as Rat Heart B. </div></div>

[STEPS]
TITLE: Use of tracer dyes to label neural projections to lower urinary tract organs
AUTHORS: Janet Keast, Peregrine Osborne

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is used to visualise sensory and autonomic neurons innervating the bladder body (dome), bladder trigone or proximal urethra in an experimental adult male or female rat. The protocol is performed under anesthesia and should incorporate all local requirements for standards of animal experimentation, including methods of anesthesia, surgical environment, and post-operative monitoring and care.</div></div>

[STEPS]
?. [Preparation for surgery]
Prepare tracer dye solutions: Fluorogold or Fast Blue (each 2% w/v in sterile water).
?. [Preparation for surgery]
Anesthetise animal (2.5% isoflurane in oxygen, or as required for maintenance)
?. [Preparation for surgery]
Apply eye lubricant and place animal on heated pad.
?. [Preparation for surgery]
Shave and clean the ventral abdomen.
?. [Surgery]
Perform a midline incision in the skin and then the muscle, then gently move organs to visualise the required injection site.
?. [Surgery]
Microinject sterile tracer solution at the selected injection site using a Hamilton Neuros Syringe attached to a 33G needle. At each injection site, hold the needle in place for ~5 seconds after ejection of the dye, to enable the dye to spread to the underlying tissue. This also minimises leakage.
Injections into the bladder body (dome) are made bilaterally on dorsal and ventral aspects; total volume ~ 5 µl. Single injections are made into the bladder trigone or dorsal aspect of the proximal urethra (maximum 0.3 µl per injection).
?. [Surgery]
Wash all injection sites with sterile saline.
?. [Surgery]
Close the muscle and skin using approved procedures. Administer analgesics and monitor animal during postoperative period as per local approved procedure.
?. [Tissue harvesting]
To analyse tracer dye distribution in ganglia or the injection site, 5-14 days after surgery, fix animals by intra-cardiac perfusion, then remove tissues of interest for further study.
For studies of the lower urinary tract innervation, these tissues would typically include dorsal root ganglia (DRG) from L1, L2, L6 and S1 spinal levels and the pelvic ganglia (synonym, major pelvic ganglia). It is also recommended that the lower urinary tract tissues are also removed for microscopic validation of the tracer injection site.
TITLE: Chemically Labile Linkers
AUTHORS: BOC      Sciences

[DESCRIPTION]
The chemically labile linkers, which include acid-cleavable linkers and disulfide linkers, are extensively applied to ADCs due to their ability to undergo fracture, increasing the acidity of the endosomal-lysosomal pathway, as well as the concentration of glutathione inside cells. BOC Scienceshas developed highly integrated approaches that can conjugate antibodies and payload via a linker to compose ADC-related products quickly. We are capable of completing small (mg) and large (g) quantities of ADC small molecule products and transferring these technologies to a bulk manufacturing plant. To ensure quality, all final products will be thoroughly analyzed by our advanced technologies, including mass spectrometry and chromatography.

[STEPS]
TITLE: DNA Ethanol Precipitation (SOP009.v1.1)
AUTHORS: Rory  Kruithoff, Douglas  Shepherd

[DESCRIPTION]
Document Summary: This document, SOP002 - DNA Ethanol Precipitation, describes a method to concentrate or dry DNA using ethanol to reduce the solubility of dissolved DNA causing it to precipitate out of solution.  Alternatively, a vacuum concentrator can be used if one is available. 

[BEFORE_START]
Quick Overview:

Step 1 – Precipitate DNA
Step 2 – Pellet DNA and dry
Step 3 – Resuspend DNA

v1.1 revision notes
Updated SOP to standard formatting
Updated document description

[STEPS]
SECTION: Precipitate DNA
1. Add  1/10 volume of 3 M Na-Acetate pH 5.2, and 2 to 2.5 volumes of ice-cold 100% ethanol to the DNA sample.

SECTION: Precipitate DNA
2. Mix, and store at -20 °C for at least 60 min to precipitate the DNA.  Typically, DNA will be left at -20 °C 60 minbefore proceeding to part 2.

SECTION: Pellet DNA and dry
3. Recover the precipitated DNA by centrifugation at full speed in a microcentrifuge for 15 min- 20 min to form a pellet.

SECTION: Pellet DNA and dry
4. Pour off the ethanol and wash the pellet twice with -20 Room temperature70 % (v/v).
SECTION: Pellet DNA and dry
5. Allow the DNA pellet to air-dry.

SECTION: Resuspend DNA
6. Re-suspend the DNA in a suitable volume of sterile TE buffer or nuclease-free water.  
SECTION: Resuspend DNA
7. Store at -20 °C and avoid any unnecessary freeze-thaw cycles.
TITLE: Nuclei Isolation for Human Ovary Explants
AUTHORS: Nicolas Martin

[DESCRIPTION]
This protocol uses fresh frozen human ovary explants to isolate nuclei suspension.

[GUIDELINES]
This protocol needs prior approval by the users' institutional review board (IRB) or equivalent ethics committee(s).

[STEPS]
SECTION: Nuclei Isolation Protocol for Human Ovary Explants
1. Chapter 1—Single Cell Gene Expression & Chromium Fixed RNA Profiling of the protocol CG000505 REV A was used to isolate nuclei from frozen human ovary explants with the following modifications: 1) a cordless motor pestle (VWR, Catalog number 47747-370) was used for Step f, Page 30 and 2) the samples were incubated for 15 min on ice for Step h, Page 30. 

https://www.10xgenomics.com/support/single-cell-gene-expression/documentation/steps/sample-prep/chromium-nuclei-isolation-kit-sample-prep-user-guide