eamag/protocols-with-steps huggingface dataset:
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol describes the transfection of mammalian cells with <em>in vitro</em> transcribed, unmodified, non-charged tRNAs. We have used this method successfully to raise intrinsic tRNA concentrations in HeLa<sup>1</sup> and N2a cells<sup>2</sup>. Furthermore, tRNAs synthesised as described in this protocol have been shown to be translation competent<sup>1</sup>.</p>
<p> </p>
<p>References</p>
<p>1. Kirchner <em>et al</em>. Alteration of Protein Function by a Silent Polymorphism Linked to tRNA Abundance. <em>PLoS Biology. </em>2017, <em>in press.</em></p>
<p><em>2. </em>Girstmair <em>et al</em>. Depletion of Cognate Charged Transfer RNA Causes Translational Frameshifting within the Expanded CAG Stretch in Huntingtin. <em>Cell Reports</em>. 2013, 3(1):148-59. DOI: <a href="http://dx.doi.org/10.1016/j.celrep.2012.12.019" target="_blank">http://dx.doi.org/10.1016/j.celrep.2012.12.019</a></p>

[BEFORE_START]
<p>Considerations for tDNA oligo design:</p>
<ol>
<li>Single stranded tDNA oligonucleotides encoding the desired tRNA sequence and the T7 promoter sequence (5'-TAATACGACTCACTATAG-3') are annealed and the resulting 5' overhangs are subsequently filled-up in order to create a linear dsDNA molecule. This dsDNA molecule then serves as template for the subsequent tRNA transcription, whereby the T7 polymerase then starts transcription at the last base of the T7 promoter (i.e. G) and then transcribes 5' → 3' using the opposite strand as a template. Hence, the last nucleotide of the T7 promoter sequence (i.e. G) will be the first base of the synthesised tRNA. It should be noted that therefore only tRNAs starting with a G as first base can be transcribed using the T7 promoter.</li>
<li>The length of the overlapping parts between 5' and 3' tDNA oligonucleotides should be approx. 20 nucleotides.</li>
<li>Example of human tRNA<sup>Thr</sup>(UGC) tDNA oligonucleotide:<br /><em>Full-length tRNA<sup>Thr</sup>(UGC)</em><br />GGCGCGGTGGCCAAGTGGTAAGGCGTCGGTCTCGTAAACCGAAGATCACGGGTTCGAACCCCGTCCGTGCCTcca<br />+ T7 promoter<br /><strong>TAATACGACTCACTATAG</strong>GCGCGGTGGCCAAGTGGTAAGGCGTCGGTCTCGTAAACCGAAGATCACGGGTTCGAACCCCGTCCGTGCCTcca<br /><em>Fwd tDNA oligonucleotide</em><br /><strong>TAATACGACTCACTATAG</strong>GCGCGGTGGCCAAGTGGTAAGGCGTCGGTCTCGTAAACC<br /><em>Rev tDNA oligonucleotide (reverse complement)</em><br />tggAGGCACGGACGGGGTTCGAACCCGTGATCTTCGGTTTACGAGACCGACGCCT<br /><br /></li>
</ol>

[STEPS]
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TITLE: Bioanalyzer Operation for IVT Products
AUTHORS: Allen Institute for Brain Science

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is used to operate the Bioanalyzer which is used as a quality control step for IVT products. Peak size, shape, and sample concentration are recorded and used to evaluate samples on a pass/fail level.</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 1U19MH114830-01. 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: Smell discrimination test
AUTHORS: daniel.dautan daniel, Per Svenningsson

[DESCRIPTION]
Behavioral test to assess smell discrimination in mice. 

[STEPS]
1. Transfer the cage into the experimental room at least 1h before testing. Always maintain the cage in a ventilated cage rack to avoid smell contamination.
2. Set the open field before starting experiment. The open field is a dark-grey box of 40 x 40 cm with clean floor and a light suspended above the center. A maximum of 150 lux is display to avoid excessive stress. The experiment need to be done in a highly ventilated room.
3. At a randomly selected corner, place the 8cm plexiglass cylinder which has holes on the side.
4. Always start with the positive smell as the TMT smell might affect further experiments.
5. Before the test place ~10 chocolate flavor pellets (Bio Serv FO5301) into the plexiglass cylinder.
6. Transfer the mice into the center of the open field using a paper tube.
7. Record the mice's positions for 20 minutes using tracking software, such as Ethovision XT or AnyMaze.
8. At the end of the test, remove the mice from the apparatus. Remove the cylinder and clean the apparatus with 50% ethanol before proceeding to the next animals.
9. On the following day, repeat the experiment but this time the cylinder needs to be placed in a different corner.
10. In the cylinder place a cotton ball that was covered with ~20µl of 5% TMT solution (Sigma Aldrich 13623-11-5), in water.
11. Place the mice in the center of the open field and record positions for 20 min.
12. Clean thoroughly the cage after each experiment with 50% ethanol.
13. Using the tracking software extract:
a) distance travelled for 20 min
b) average speed
c) time in the center of the apparatus
d) time in the corner paired with the smell
14. Using each value, compare the time spent in chocolate or TMT corner and the ratio correspond to the olfactory discrimination index.
TITLE: Mouse Ovariectomy
AUTHORS: Sabina Marciano, Roberta Marongiu

[DESCRIPTION]
This protocol outlines the steps to perform an Ovariectomy in a mouse

[STEPS]
1. Anesthetize the mouse with Ketamine ( 100 µL / 100 g )
2. Apply eye ointment
3. Shave and disinfect the area below the ribs
4. Disinfect the surgical tools
5. Place the mouse on the side

 
6. Align 2 tweezers with the arms/legs and use the crossing point as a reference for the
incision
  

7. Perform a small incision and pull out the fat/ovary

  

8. Clamp right below and above the ovary

  

9. Remove the ovary
  

10. Suture both the inner and out layers
11. Disinfect the suture with antibiotic ointment
TITLE: X-region PolyU/UC mutagenesis
AUTHORS: Carolina Lopez

[DESCRIPTION]
Protocol to PCR and in vitro transcribe the HCV X region w/out mutagenesis

[STEPS]
SECTION: HCV X-region PolyU/UC PCR - T7 in vitro transcription
1. PCR:
1) Thaw component and cDNA, spin down before use
2) Assemble reaction: Master Mix:  10X (each reaction assemble 3 tubes)
 
              Total: 50 µL

3) Run PCR  
 

SECTION: II.	In vitro transcription:
2. ➢	Spin-down all reagents 
➢	Keep the 10X Reaction Buffer at room temperature while assembling the reaction. 
➢	Vortex all solutions until they are completely in solution. 
➢	Assemble dNTP mix (2μL ATP+ 2μL CTP+ 2μL GTP+ 2μL TTP/reaction).
➢	Store the ribonucleotides on ice!

Assemble the reaction in an appropriate RNase free PCR tube.
1)	1 µg DNA plasmid (digested)
2)	8 µL NTP Mix
3)	2 µL 10X Reaction Buffer
4)	0.5-1 µL RNaseOUT (RNase Inhibitor)
5)	2 µL T7 enzyme mix
6)     RNase Free water up to 20 µL

•	Pipette the mixture up and down gently and microfuge 
•	Incubate 3-6hrs at 37 °C using PCR machine. The shorter the ivtRNA, the longer the reaction time needed.
•	(Optional but do most times) Template plasmid Digestion: 1μLTurbo DNase and mix well, 15 min at 37 °C
SECTION: IV.	RNA precipitation:  Transfer to an 1.5ml RNase free vial
3. If you expect very low yields of RNA, do not dilute the transcription reaction with water prior to adding the LiCl.

•	Add	30 µL H2O
	        30 µL LiCl 
•	Mix well, incubate 30 min at -20 °C
•	Centrifuge at 4°C/20 min/max
•	Remove supernatant
•	Add 1 mL 70% COLD ethanol
•	Centrifuge at 4°C/5 min/max
•	Remove supernatant and allow the pellet to dry
•	Resuspend in 15-20 µL RNase-free H2O
•	Place the tube 5 min at 65 °C water bath
•	Dilute 1 µL in 19 µL H2O to measure concentration
TITLE: Optimization of Fragmentation Temperature
AUTHORS: Dakota Betz

[DESCRIPTION]
The standard fragmentation temperature is 37°C. If you are fragmenting high-quality genomic DNA, any other
high-complexity DNA sample, or FFPE DNA to a mode fragment length <500 bp, it is unlikely that you will have to
change or optimize the fragmentation temperature. 

Low-complexity samples (e.g., small viral genomes, plasmids, long amplicons and cDNA) may, however, be
over-fragmented at 37°C, even with short incubation times. The likelihood of over-fragmentation depends on
the nature, molecular weight/length of the input DNA, the desired size distribution after fragmentation and, to a lesser
degree, the DNA input into fragmentation. For example,  100 ng of a 1.8 kb PCR product will yield a similar mode
fragment length (~300 bp) as 100 ng E. coli or human genomic DNA when fragmented at 37°C for 10 min,
whereas 1 ng of a 1 kb PCR product will be fragmented to a mode size <250 bp using the same parameters.

[BEFORE_START]
Shipping and Storage
The enzymes provided in this kit are temperature sensitive, and appropriate care should be taken during shipping and
storage. KAPA HyperPlus Kits are shipped on dry ice or ice packs, depending upon country of destination. Upon
receipt, immediately store enzymes and reaction buffers at -15°C to -25°C in a constant-temperature freezer. When
stored under these conditions and handled correctly, the kit components will retain full activity until the expiry date
indicated on the kit label.

Handling
Always ensure that KAPA HyperPlus Kit components have been fully thawed and thoroughly mixed before use.
The End Repair & A-Tailing Buffer and Ligation Buffer may contain precipitates when thawed at 2°C to 8°C. These
buffers must be thawed at room temperature and vortexed thoroughly before use. KAPA HiFi HotStart ReadyMix (2X)
contains isostabilizers and may not freeze completely, even when stored at -15°C to -25°C. Nevertheless, always
ensure that the ReadyMix is fully thawed and thoroughly mixed before use. Reaction master mixes prepared from
the enzymes and buffers for fragmentation, end repair and A-tailing, as well as for ligation, are very viscous and
require special attention during pipetting. Keep all enzyme components and master mixes on ice as long as possible
during handling and preparation.

Quality Control
All kit components are subjected to stringent functional quality control, are free of detectable contaminating exoand
endonuclease activities, and meet strict requirements with respect to DNA contamination.

[STEPS]
1. To determine the optimal fragmentation parameters for low-complexity samples, or high-complexity samples
when the desired mode fragment length is >500 bp:

1.1. Set up four replicate reactions with a non-precious, bulk sample that is representative of the actual samples
to be processed.
1.2. Fragment two of the samples at 37 °C, for 5 min and10 min, respectively. Repeat these fragmentations with the other two samples, but at 30 °C.

1.3. Complete the library construction process, and  evaluate the size distribution of the final libraries
electrophoretically.
TITLE: Post Patch Clamp Slice Fixation
AUTHORS: Allen Institute for Brain Science

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes the process to fix recorded/filled cells from mouse and human brain slices with 4% PFA/2.5% Glutaraldehyde in PBS for future histochemical processing.</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: ToLA Assembly Pipeline 1
AUTHORS: Marcela  Uliano-Silva, Ksenia  Krasheninnikova, Shane  A. McCarthy

[DESCRIPTION]
Here you find the first assembly pipeline developed by the Tree of Life Assembly Team (ToLA) to assemble the genomes sequenced in the first 2 years of the Darwin Tree of Life Project (DToL), which belongs to the Tree of Life Department of the Wellcome Sanger Institute (https://www.darwintreeoflife.org). DToL aims to generate high-quality genomes for all named eukaryotes occurring in Britain and Ireland. This protocol describes all softwares and bioinformatics steps (assembly, polishing and scaffolding) ran to generate chromosome-level genomes with three data types: (i) PacBio Hifi, (ii) Linked-reads (Chromium 10X) and (iii) Hi-C. First, PacBio HiFi reads are checked and cleaned for adaptors ....

 

[STEPS]
SECTION: Reads trimming
1. Pacbio Hifi reads trimming

Input your Pacbio Hifi reads to HiFiAdapterFilt (https://github.com/sheinasim/HiFiAdapterFilt)

HiFiAdapterFilt will convert .bam to .fastq and remove reads with remnant PacBio adapter sequences.
TITLE: Locomotion test for mice 
AUTHORS: Csilla Novák, Andrés Mauricio Jaramillo Flautero, Matthias Prigge

[DESCRIPTION]
The locomotion test is an open field test in darkness, so it does not aim to measure anxiety, only locomotor activity.

[STEPS]
SECTION: Behaviour
1. Transport the mice to the behaviour room and put the mice in individual cages.
SECTION: Behaviour
2. Leave them in the lobby in darkness or with red light for at least 30 minutes.
SECTION: Behaviour
3. Turn on the red lights in the room.
SECTION: Behaviour
4. Place a 50x50 cm arena on the table.
SECTION: Behaviour
5. Clean the arena and the table with 70% ethanol.
SECTION: Behaviour
6. Turn on the infrared lights below the arena. Open Pylon Viewer (see settings below), and make sure that you get a good image quality.
SECTION: Behaviour
7. Bring a mouse to the behaviour room. Open the cage and gently get the animal out. Do not chase it around in the cage.
SECTION: Behaviour
8. Place the animal in the center of the arena. Depending on your handling protocol, you can put it in by the tail or in the palm of your hand.
SECTION: Behaviour
9. Make sure to start the recording at the moment when you place the mouse in the arena.
SECTION: Behaviour
10. Make a recording of 30 min. The recording will be automatically saved; make sure to rename it.
SECTION: Behaviour
11. Pick the mouse out of the arena and put it back in the cage.
SECTION: Behaviour
12. Count poop pellets and take a note of them.
SECTION: Behaviour
13. Clean the arena and the table with 70% ethanol. If you feel the necessity, you can rinse the area with distilled water.
SECTION: Pylon Viewer
14. Connect the camera to the computer and to a power source.
SECTION: Pylon Viewer
15. Open the Pylon Viewer and select the camera.
SECTION: Pylon Viewer
16. At AOI controls, crop the image, so that you only see the setup. You can only change the width and the height of the image when there is no continuous shot or recording.
SECTION: Pylon Viewer
17. With the help of a test mouse, you can set the image quality. On the camera, you can set the aperture and the focus. In Pylon Viewer, you can play around with Analog Controls > Gain (Raw), Black level (Raw), and Acquisition Controls > Exposure time (Raw).
SECTION: Pylon Viewer
18. Go to Window and select Recording Settings. Set the Output format to .mp4, the fixed playback speed to 67 fps, and the Recording buffer size to 100 frames. Record a frame every 15 milliseconds. Stop the recording after 30 min. Set the output folder.
SECTION: Data analysis
19. Tracking of animal kinematics most easily done with DLC (link)

To identify individual behavior motifs we use VAME (link) or A-Soid (link)
TITLE: Single cell isolation and monoclonal culture establishment of Acanthamoeba castellanii using migration on agar plates
AUTHORS: Morgan Colp

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Isolation of single </span><span style = "font-style:italic;">Acanthamoeba castellanii </span><span>cells from agar plates for establishment of monoclonal cultures. This protocol can be useful for isolating clones of a mixed wild-type population or of transformants. After isolation, growth can be re-established in typical Neff liquid medium. </span></div></div>

[STEPS]
?. [E. coli preparation]
Grow 24 hour culture of E. coli K-12 in LB
?. [E. coli preparation]
Pellet E. coli and resuspend in an equal volume of water or Page's Amoeba Saline in 15 mL Falcon tube
?. [Plate set-up]
Pour PAS agar into several (3 to 5) Petri plates per 10 cells planned to isolate and let set.
?. [Plate set-up]
Spread 1 mL aliquot of heat-killed E. coli onto each plate and continue spreading until dry.
?. [E. coli preparation]
Heat-kill E. coli in 65 degree C water bath for 20 minutes.
?. [Plate set-up]
Scrape the internal surface of a 7 to 14 day old Acanthamoeba castellanii liquid culture and gently shake the flask to suspend cells.
?. [Plate set-up]
Pipette 1 microlitre of Acanthamoeba suspension into the centre of each plate.
?. [Cell isolation]
After 3 to 5 days, cells will have migrated far enough from the centre of the plate that the furthest ones may be 1 or more centimetres apart. Check this with an inverted microscope to determine when this is the case. There will be visible trails through the bacterial lawn to see where cells have migrated.
?. [Cell isolation]
When cells have dispersed enough (1 cm or more apart), cut 1 cm squares out of the agar, containing only one cell each (not too difficult to see with an inverted microscope) and place one square into each well of a 12 well culture plate. You can cut squares from the middle of the plate into a few wells as positive controls.
?. [Cell isolation]
Add enough PYG medium to fully cover the agar cubes (you can fully fill these wells) and allow growth at room temperature. Growth can be observed in these plates under an inverted microscope. When cells start to cover the entire area of the bottom of a well (at the same density you might see from 4-5 days growth in regular culture) you can scrape those wells and transfer the entire contents back into flasks for growth in typical culture conditions. 10 ug/ml polymyxin B can be added to control bacterial growth in case not all E. coli were heat killed. If you are isolating single transformed cells you can select immediately or wait a few days to allow your new monoclonal populations to establish in your flasks.
TITLE:      ELISA for quantification of IL-29 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-29 is a type-3 interferon and produced by virus-infected cells, dendritic cells, and regulatory T-cells. It upregulates viral protective responses. Virus-infected cells may regulate IL-29 genome. </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-29 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-29 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-29 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-29 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-29 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-29 standard dilutions and the human IL-29 sample concentration is determined.
?. For better results place the microplate on a microplate shaker in every incubation.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Protocol for performing ATAC-seq on nuclei isolated from <em>Drosophila melanogaster</em> stage 5 embryos that were flash frozen and then cut in half along the anterior-posterior midline. Data from this protocol are presented in the following paper: https://www.biorxiv.org/content/early/2017/09/27/195073.</p>
<p> </p>
<p>This protocol is based adapted from the following sources:  https://www.nature.com/nmeth/journal/v10/n12/full/nmeth.2688.html and http://journals.plos.org/plosgenetics/article?</p>

[STEPS]
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TITLE: Fluorescent Western Protocol
AUTHORS: Steven J Burgess, Lynn    Doran

[DESCRIPTION]
Analysis of proteins using fluorescent immunoblot.  

Note:  
- The choice of secondary antibody depends on the choice of primary antibody, whether it is derived from a mouse (monoclonal) or a rabbit (polyclonal).  
- It is advisable to stick to the 800CW wavelength to avoid problems with chlorophyll autofluorescence encountered with the 680CW antibodies.  

Literature: 
Licor's "Fluorescent Western Blot Detection"
Licor's "Good Westerns Gone Bad"

[BEFORE_START]
Isolate total protein via Leaf Protein Extraction for Immunoblot (Soybean, Cowpea, Tobacco).
Quantify protein via Protein Concentration Determination using Qubit 4 Fluorometer.
Separate protein components via SDS-PAGE gel electrophoresis.
Transfer protein to a membrane via Protein Transfer using Bio-rad TransBlot Turbo.

[STEPS]
1. 
Keep membranes in the black Western Blot incubation box for all steps, this is important after adding the secondary antibody because the signal is light-sensitive and will become bleached if not exposed to light for a long enough period.

 


2. Wet with 1x PBS for 2 min min

3. Rinse membrane with dH2O
4. Discard PBS and incubate with   60 min  at Room temperature
 



6. Incubate with primary antibody (appropriate dilution in    at 4 °C    (in cold room) with gentle agitation on a platform shaker   .
 


5. Prepare 1 L PBS-T solution by diluting 100 mL of 10X PBS

7. Pour off the primary antibody and rinse the membrane with PBS-T.
 

8. Cover the membrane with PBS-T, shake vigorously on a platform shaker at  . Repeat 3 times.

 


9. Create a working dilution of secondary antibody using . . For PVDF membranes only, add 0.01% SDS to the antibody diluent.    Microcentrifuge secondary antibody and pipette from supernatant to precipitate out any protein complexes that may have formed during storage.  

 

10. Incubate for 60 min Room temperature with gentle agitation on a platform shaker.
 

11. Pour off the secondary antibody and rinse membrane with distilled water to remove residual blocking agent.
12. Cover the membrane with PBS-T, agitate  
 

13. Discard PBS-T. Repeat step 12  three times.
 

14. Rinse then cover the membrane with 1x PBS,  . 

15. Proceed to imaging blot on LI-COR Odyssey CLx imaging system