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

[DESCRIPTION]
<p>Here we describe a protocol for generating 3D human intestinal tissues (called organoids) in vitro from humanpluripotent stem cells (hPSCs). To generate intestinal organoids, pluripotent stem cells are first differentiated into FOXA2(+)SOX17(+) endoderm by treating the cells with activin A for 3 d. After endoderm induction, the pluripotent stem cells are patterned into CDX2(+) mid- and hindgut tissue using FGF4 and WNT3a. During this patterning step, 3D mid- or hindgut spheroids bud from the monolayer epithelium attached to the tissue culture dish. The 3D spheroids are further cultured in Matrigel along with prointestinal growth factors, and they proliferate and expand over 1-3 months to give rise to intestinal tissue, complete with intestinal mesenchyme and epithelium comprising all of the major intestinal cell types. To date, this is the only method for efficiently directing the differentiation of hPSCs into 3D human intestinal tissue in vitro.</p>

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Dengan melakukan import data teman-teman sesama peserta ke Gmail/Google Contacts, maka Anda dapat dengan mudah menghubungi teman-teman peserta lainnya, melalui telepon, SMS, WhatsApp, Telegram, maupun email.</p>
<p> </p>
<p>File yang dibutuhkan:</p>
<ol>
<li>File CSV Google Contacts - ada di Dropbox bersama</li>
</ol>

[STEPS]
?. 
?. 
?.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Calibration procedure for functionally aligning inertial sensors fixed to the shanks, thighs, and trunk (e.g. sacrum, sternum). The movements were originally designed for analyzing skiing movements where the person is wearing ski boots. However, the same protocol can be applyied also barefoot or with regular shoes.</p>
<p> </p>
<p>Please refer to the protocol's guidelines for more information how to obtain a sample data set and the matlab code needed to process the functional calibration.</p>

[BEFORE_START]
<p>Place the inertial sensors on the shanks, thighs, and trunk (e.g. sacrum, sternum). The exact sensor orientation does not matter, i.e. they don't need to be put in the exact same way for each new experiment. However, the sensors should be placed such as to reduce soft tissue artefacts. A good example of soft tissue artefacts of the thigh can be seen at the beginning of the functional calibration for the hip ad/abduction movement. Activation of the thigh muscles at the start of the movement are moving the inertial sensor visibly upwards.</p>
<p>For example, a good location for the shank sensors is the tibial plateau, about 10-15cm below the knee, slightly medially.</p>

[GUIDELINES]
<ul>
<li>Before each movement, the person should remain motionless for 1-2 seconds to allow better data segmentation.</li>
<li>Trunk rotation: ski poles are only needed to ensure a better control of the rotation axis. The movement can also be performed with the hands held behind the head.</li>
<li>Hip ad/abduction: ski poles are only needed for balance. The movement can be performed without ski poles but requires quite good balance control abilities of the person.</li>
</ul>
<p> </p>
<p><em><strong>The code to analyze the data and the data from the functional calibration filmed from the lateral view has been posted on Code Ocean, under the DOI 10.24433/CO.3f699198-4e77-4d51-8482-13d1b9ad93b8<br /></strong></em></p>

[STEPS]
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TITLE: SPARC_Duke_PelotGrill_OT2-OD025340_PigVagusNerve_Morphology
AUTHORS: Nicole A. Pelot, Gabriel B. Goldhagen, Jake E. Cariello, Warren M. Grill

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The protocol describes image segmentation and image analysis methods to quantify pig vagus nerve morphology from Masson's trichrome-stained nerve cross sections.</div></div>

[STEPS]
?. [Image Segmentation]
We used Nikon's NIS Elements software (v5.02.01, Build 1270) to segment pig vagus nerve micrographs stained with Masson's trichrome using the General Analysis RGB tool.
?. [Image Segmentation]
We converted the binary segmented image into “Graticule Masks”, binary images saved as TIFs.
?. [Image Analysis]
We imported the TIFs into Matlab and generated a data structure of the x and y coordinates of the pixels for each closed boundary of the loaded binary images using the bwboundaries function.
?. [Image Analysis]
We scaled the pixel coordinates to microns using the segmented scale bar.
?. [Image Segmentation]
For each image, we selected preprocessing steps, such as smoothing and sharpening.
?. [Image Segmentation]
For each image, we selected ranges of hues, saturations, and intensities to values that identify the endoneurium (fascicles) and different values to identify the entirety of the nerve.
?. [Image Segmentation]
For each image, we selected postprocessing steps, such as setting a minimum size criterion (eliminate small off-target regions), smoothing, cleaning, closing, and filling holes.
?. [Image Segmentation]
We made manual adjustments as needed, including manual deletion of off-target regions and filling of target areas that had not been captured.
?. [Image Analysis]
We calculated cross-sectional area of each fascicle and nerve using Matlab’s polyarea. Effective diameter (for a nerve or fascicle) is the diameter of the circle that has the same cross-sectional area as the raw trace.
TITLE: PNA-lipophilic Ligand Conjugates Synthesis
AUTHORS: Cathy  Miller

[DESCRIPTION]
Lipophilic ligands are a class of substances suitable for cell transport, they are lipids with a certain hydrophilic-lipophilic balance. When transported across the cell membrane, the hydrophilic end faces the extracellular water-soluble environment, and the hydrophobic end faces the cell membrane. A typical representative of this modification is the covalent attachment of bile acid and cholesterol to PNA. The conjugate also has a certain liver-targeting effect in vivo and has the potential to develop into a liver-targeted preparation.

The covalent binding of PNA with hydrophobic compounds or lipids has some excellent properties. For example, by connecting a hydrophobic long carbon chain to the N-terminus of PNA and amino acid residues to the C-terminus, the conjugate has good hydrophilicity and lipophilicity, and shows better specificity when hybridizing with DNA. The conjugate can also self-assemble into spherical micelles, and has application potential in biosensors, gene drugs, etc. PNA-lipophilic ligand conjugate also shows good performance in cell transport.

Creative Peptideshas completed many PNA conjugate synthesis cases. Based on these projects, our team has accumulated a deep understanding of product requirements. With rich experience and scientific knowledge, our experts are dedicated to helping our customers navigate and accelerate your PNA synthesis plan. By combining scientific knowledge and laboratory experience, we are committed to expanding your understanding and applications of PNA conjugates. Pleasecontact us to learn more.

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Luria Broth (LB) is a nutrient-rich liquid media that is used to grow bacterial cultures. Luria Broth consists of Tryptone, Yeast extract and Sodium Chloride mixed into distilled water.</p>

[BEFORE_START]
<p>Give yourself about 2.5 hours to finish making the sterilized, ready-to-use LB broth. Making the LB broth doesn't take long, but the autoclave use can take up to 2.5 hours.</p>

[GUIDELINES]
<p><strong>Equipment needed:</strong></p>
<p>- 500 mL or 1-liter bottle</p>
<p>- graduated cylinder (100mL or greater)</p>
<p>- scale for weighing</p>
<p>- Autoclave</p>
<p>- autoclave gloves</p>

[STEPS]
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TITLE: Ancestral State Reconstruction
AUTHORS: Dakota Betz

[DESCRIPTION]
Our protocols are constantly evolving and old versions will be deleted.
The documents here are not intended to be cited in publications

[STEPS]
SECTION: Programs and Dependencies
1. Mesquite
Download: https://www.mesquiteproject.org/Installation.html

RAxML-ng
Download: https://github.com/amkozlov/raxml-ng
GUI 2.0 (compatible with ng): https://antonellilab.github.io/raxmlGUI/

FigTree


Text Editor (options below)
SublimeText:  https://www.sublimetext.com/3
BBEdit: https://www.barebones.com/products/bbedit/download.html

 

SECTION: Tree Import
2. Tree import into Mesquite works best if the tree file is already in nexus or newick format. If your tree is not already in this format, open it in FigTree and select Export Trees, then choose either Nexus or Newick format and check the box to Save as currently displayed if applicable. 
SECTION: Tree Import
3. To import your tree into Mesquite, open Mesquite (.jar version works best). Select File > Open and choose your tree file.
SECTION: Matrix Set-up
4. On the left side menu under Project, select Add... > New Character Matrix. This should autofill with the taxon names from your tree. Type in the number of characters you need to code for your ancestral state reconstruction and select Categorical Data.
SECTION: Matrix Set-up
5. Once the matrix is created, fill in the character labels at the top. Select Matrix > Show Matrix Info Panel. This should produce a tab on the right labeled "State Names". You can type in the character state names/labels that you'd like for your characters here (e.g. if your character is "habitat", you can add in "vent, seep, organic fall" as character states). Keep track of the numbers that apply to each state so you know which ones to enter for each of your taxa in the matrix.
 

SECTION: Ancestral State Reconstruction
6. Once your matrix is filled out, there are various reconstruction options. The most frequently used in our lab are parsimony reconstructions (no model applied) and likelihood ancestral states (applies a model). We'll do the latter here.
SECTION: Ancestral State Reconstruction
7. Return to the tree Window. Select Analysis:Tree > Trace Character History and select your reconstruction method: likelihood ancestral states. You can also select this from the Method submenu if you have already selected Trace Character History once and want to repeat your analysis.
SECTION: Viewing the Results
8. Use the "balls and sticks" style to view your resulting tree. Select Display > Tree Form > Balls & Sticks
SECTION: Viewing the Results
9. You may also want to see relative likelihoods and branch lengths. Select Display > Branches proportional to lengths to see a tree with branchlengths (note that these must have been present in the tree you imported to begin with in order to display them). Select Drawing Menu > Tree Form with line style "square" to see relative likelihoods and branch lengths.
TITLE: Promega BacTiter-Glo Assay
AUTHORS: NUS iGEM

[STEPS]
?. Prepare BacTiter-Glo reagent mix by transferring BacTiter-Glo buffer into BacTiter-Glo substrate
These reagents are part of a set - Promega's BacTiter-Glo Assay.
?. Aliquot  of cells into two wells of 96-well plate (duplicates)
50 µl
?. Add  of BacTiter Glo reagent mix into each well
50 µl
?. Include PBS-only wells as blank
?. Measure luminescence using microplate reader
TITLE: Buck Institute Morphology H &amp; E staining protocol
AUTHORS: Stella Breslin, Akos A Gerencser

[DESCRIPTION]
Histological Stain for FFPE slides
RESULTS:  Nuclei-blue; Cytoplasm, red blood cells and connective tissue-shades of pink

[BEFORE_START]
Slides can be placed in 60 C oven prior to deparaffinization. 

[STEPS]
SECTION: Hematoxylin and Eosin Staining
1. Deparaffinization and rehydration: 
2 x xylene 7 min, 
100% ethanol 4 min, 
95% 4 min, 
80% 4 min, 
70% 4 min, 
bring to water.
SECTION: Hematoxylin and Eosin Staining
2. Hematoxylin staining for 30 seconds - 4 min. using filtered dye (usually 2 mins) - Modified Mayes’s Hematoxylin HXMMHPT StatLab Medical Products
SECTION: Hematoxylin and Eosin Staining
3. Rinse in ddH20 x2
SECTION: Hematoxylin and Eosin Staining
4. 10% acetic acid rinse (acid into water) – 1 min
SECTION: Hematoxylin and Eosin Staining
5. rinse in ddH20 x2
SECTION: Hematoxylin and Eosin Staining
6. TBS (1x) rinse or Scott’s Tap Water (1L + 3.5g Sodium Bicarb + 20g Magnesium Sulfate pH 8.0)  - 2 mins
SECTION: Hematoxylin and Eosin Staining
7. Rinse in ddH20
SECTION: Hematoxylin and Eosin Staining
8. Rinse in 70% ETOH - 1 min
SECTION: Hematoxylin and Eosin Staining
9. Stain Eosin for 0.5 to 2 minutes (keep an eye on intensity of pink) – Eosin Y Stain STE0150 StatLab Medical Products
SECTION: Hematoxylin and Eosin Staining
10. Rinse in 90% EtOH
SECTION: Hematoxylin and Eosin Staining
11.  Rinse in 
2 x 95% EtOH, 
2 x 100% EtOH,  
xylene
SECTION: Hematoxylin and Eosin Staining
12. Coverslip with Permount Mounting Medium EMS #17986-01
TITLE: Link Github to Puhti
AUTHORS: Alise Ponsero

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">How to connect your github account to a local machine or an HPC cluster (example at Puhti CSC)</div></div>

[STEPS]
?. [Verify that git is installed]
Open a terminal and log onto the remote machine (if applicable).In order to quickly test that Git is installed on the machine type:git --helpYou should have the git usage printed out!
?. [Configuration of Github]
To configure the Git account type:git config --global user.name "Your name here"git config --global user.email "your_email@example.com"Replace the above code with your github username and email address.
?. [SSH keys]
id_rsaid_rsacd ~/.ssh/~/.ssh/lsDo you see any id_rsa and id_rsa.pub files?
?. [SSH keys]
Then log into your Github account and open your profile settings.
?. [SSH keys]
In settings, navigate to SSH and GPG Keys
?. [SSH keys]
Finally, create a new SSH key, give it a name and paste the content of the id_rsa.pub file. Save the new key.
?. [Check the connection]
Open a new terminal window and connect to the remote machine (if applicable). Check that everything was set up properly by typing:ssh -T git@github.comYou should get the following message:
?. [Create the keys in Github]
Next, connect to your Github account and go to your profile's settings
?. [Create the keys in Github]
In Settings, navigate to SSH and GPG Keys section
?. [Create the keys in Github]
Finally create a new SSH key, give it an appropriate name and paste the content of the id_rsa.pub file.Save the key and close Github!
?. [Check the configuration]
Finally we can check if everything went well!Open a new terminal window and connect to the remote machine (if applicable). Test the configuration by typing:ssh -T git@github.comyou should see the following message:
TITLE: Preparation of TET Buffer
AUTHORS: Nicola O'Reilly, Svend Kjaer, Maria Greco

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Purpose of examination / Clinical relevance</span></div><div class = "text-block">At the end of 2019, several pneumonia cases were reported in Wuhan, China and the pathogen was confirmed as a new viral strain. World Health organization has named the newly identified coronavirus as 2019-nCoV, also known as COVID19. The disease developed into a dangerous pandemic, posing major challenges to the NHS. Although more research is necessary to better understand the virus, in response to the emergency, simple and rapid testing is essential to identify the virus in infected individuals. This will aid the implementation of efficient interventions to contain the spread, and distinguish healthcare workers who have been infected, and are required to self-isolate, from those showing similar symptoms but which are not 2019-nCoV associated. The latter category may continue to work, alleviating stress on hard-pressed healthcare resources. 2019-nCoV is an RNA virus, and the diagnostic tests detect viral RNA in swabs from patient airways using a reverse transcriptase PCR assay. Samples are submitted to HSL, an accredited reporting laboratory, and transferred to the Crick for testing.</div><div class = "text-block"><span style = "font-weight:bold;">Principles of Examination</span></div><div class = "text-block"><span>This procedure involves the preparation of TET buffer for the </span><a href="https://www.protocols.io/private/092BEFAA830211EABF5D0242AC110002" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">RNA extraction protocol</span></a><span style = ":;">.</span></div></div>

[STEPS]
?. Please select between the recipes for 2 L TET and 5 L TET.
?. Add  to a 2 L single use cell culture bottle.
[RNase free Water]
?. Add , .
[0.5 M EDTA]
?. Add , .
[1 M Tris-HCl]
?. Add .
[Tween-20]
?. Make up to 2000 ml Water (RNAse free). Swirl very well to ensure that Tween-20 is well mixed in.
?. Vacuum filter into sterile bottles.
?. Label with batch number and date.
?. Store TET buffer at solution at  ( – ).
0 Room temperature
15 °C
25 °C
?. Add  to a 5 L single use cell culture bottle.
[RNase free Water ]
?. Add , .
[0.5 M EDTA]
?. Add , .
[1 M Tris-HCl]
?. Add .
[Tween-20]
?. Make up to 5000 ml Water (RNase free). Swirl very well to ensure that Tween-20 is well mixed in.
?. Vacuum filter into sterile bottles.
?. Label with batch number and date.
?. Store TET buffer at solution at  ( – ).
0 Room temperature
15 °C
25 °C
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Use this protocol to mask <em>Symbiodinium</em> cell surface glycans prior to colonizing aposymbiotic <em>Exaiptasia</em> polyps, then measure colonization dynamics with epifluorescent microscopy.</p>

[BEFORE_START]
<p>Several variables affect symbiont uptake rate, such as how long the anemones are starved, how large they are, temperature, light, lectin concentration, and the density of symbionts used during exposure. Even keeping these constant, expect high variability during early uptake—try to use at least 5 polyps per treatment if possible. Here we present our usual approach, but adjustments may be necessary depending on the experiment.</p>

[GUIDELINES]
<p>The general strategy is to expose aposymbiotic anemone polyps to glycan-masked symbiont cells for two days, then remove the cells and allow one extra day of proliferation. Animals are fixed on the third day post exposure then symbiont density is determined through fluorescent microscopy.</p>

[STEPS]
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TITLE: CRISPR nuclease (CRISPRn) genome editing
AUTHORS: Leonardo A Parra-Rivas

[DESCRIPTION]
CRISPR nuclease (CRISPRn) genome editing

[STEPS]
1. 



LentiCRISPRv2 plasmid (RRID:Addgene_52961) was used for α-syn genome editing. All sgRNAs
were designed using CRISPick (https://portals.broadinstitute.org/gppx/crispick/public) and examined for genome-wide sequence specificityusing the National Center for Biotechnology Information’s (NCBI) Basic Local Alignment Search Tool NCBI BLAST
(RRID:SCR_004870)( http://blast.ncbi.nlm.nih.gov/Blast.cgi). The sgRNAs (Control 5’
GACGGAGGCTAAGCGTCGCAA3’ and α-syn CRISPRn 5’ GGTTCATGAAAGGACTTTCAA3’) were
cloned as described previously
 







2.   To validate the sgRNAs constructs, DIV-3 hippocampal
primary neurons were infected with lentiviruses per 6hrs (MOI=5). Transduced
neurons were cultured to maturity (DIV17-DIV21) and then lysate for western
blotting analysis and genomic analysis.
TITLE: Examination of the regression model to quantify the degree of low back pain and lower limb symptoms in patients with lumbar disc herniation by the Japanese Orthopaedic Association Back Pain Evaluation Questionnaire (JOABPEQ)
AUTHORS: Hayato Ishitani, Toshiyo Tamura, Shigehiko Kanaya

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The Japanese Orthopedic Association Back Pain Evaluation Questionnaire (JOABPEQ) was created to evaluate specific treatment outcomes in terms of physical functioning, social ability, and mental health in patients with back pain-related diseases. In this study, we investigated whether the JOABPEQ could be used to construct a regression model to quantify low back pain and lower limb symptoms in patients with lumbar disc herniation (LDH). We reviewed 114 patients with LDH scheduled to undergo surgery at our hospital. We measured the degrees of 1) lower back pain, 2) lower limb pain, and 3) lower limb numbness using the visual analog scale before the surgery. All answers and physical function data were subjected to partial least squares regression analysis. The degrees of lower back and lower limb pain could be used as a regression model from the JOABPEQ and had a significant causal relationship with them. However, the degree of lower limb numbness could not be used for the same. Based on our results, the questions of the JOABPEQ can be used to multilaterally understand the degree of lower back pain and lower limb pain in patients with LDH. However, the degree of lower limb numbness has no causal relationship, so actual measurement is essential.</div></div>

[STEPS]
TITLE: Centrifuge the whole blood to separate red blood cells
AUTHORS: George Lykotrafitis

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Centrifuge the whole blood in order to obtain the red blood cells.</div></div>

[STEPS]
?. Centrifuge the whole blood using the parameters: 500 ×g,  4 ℃, and 5 min.
?. Remove the plasma and buffy coat.
?. Add the same volume of Alsever's solution as the remaining blood components.
?. Centrifuge again using the parameters: 500 ×g,  4 ℃, and 5 min.
?. Remove the top layer.
?. Add the same volume of Alsever's solution as the remaining blood components.
?. Centrifuge a third time using the parameters: 500 ×g,  4 ℃, and 5 min.
?. Remove the top layer.
?. Add the same volume of Alsever's solution as the remaining blood component - red blood cells, and store at 4 ℃ for up to 7 days.
TITLE: RNAi Mechanism using siRNA
AUTHORS: BOC       Sciences

[DESCRIPTION]
RNA interference (RNAi) is a biological process in which RNA molecules silence specific genes, it's first discovered in plants and Caenorhabditis elegans and later in mammalian cells. This discovery is one of the most important advances in biology, so Andrew Fire and Craig C. Mello shared the 2006 Nobel Prize in Physiology or Medicine for their contribution on discovery of RNAi technology. RNAi technology is becoming an important tool for cancer research nowadays.
There are two effector molecules involving RNAi, one is small interfering RNA (siRNA for short), the other is micro RNA (miRNA for short).


[STEPS]