eamag/protocols-with-steps huggingface dataset:
TITLE: UV Crosslinking of Adherent Cells for eCLIP
AUTHORS: Eric Van Nostrand, Gene Yeo

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Profiling of RNA binding protein targets in vivo provides critical insights into the mechanistic roles they play in regulating RNA processing. The enhanced crosslinking and immunoprecipitation (eCLIP) methodology provides a framework for robust, reproducible identification of transcriptome-wide protein-RNA interactions, with dramatically improved efficiency over previous methods. Here we provide a step-by-step description for UV crosslinking of adherent cells to prepare them for eCLIP experiments.</div></div>

[STEPS]
?. [Wash Cells]
Aspirate spent media
?. [UV Crosslinking]
Place the tissue culture plate on leveled ice or a cooling block pre-chilled to 4°C
?. [UV Crosslinking]
Place the above (plate plus ice or cooling block) into the UV cross-linker.  Note: Ensure the plate is leveled.  Remove tissue culture plate lid for cross-linking.
?. [UV Crosslinking]
Transfer the cells to a 50 mL conical tube
?. [Wash Cells]
Wash the plate gently with 1x DPBS at room temperature (15 mL for a 15 cm plate).
?. [Wash Cells]
Add enough 1x DPBS to just cover the plate (5 mL for a 15 cm plate)
?. [Wash Cells]
Aspirate media
?. [Wash Cells]
Note: If all plates are at equal cell density, one plate can be sacrificed for counting – this plate would be dissociated (with trypsin, accutase, or equivalent) and cell number (per plate) counted at this stage. This is recommended for cell types that require chemical dissociation enzymes to dissociate and be properly counted, as this is not recommended post-crosslinking.
?. [UV Crosslinking]
Wash plate once with 10mL of 1x DPBS and add to the same 50 mL tube.
?. [UV Crosslinking]
While keeping the cells on ice, use a cell scraper (Corning, CLS3010-10EA) to scrape the plate.
?. [UV Crosslinking]
Cross-link at 254-nm UV with an energy setting of 400 mJoules/cm2
?. [UV Crosslinking]
Gently resuspend until the sample is homogeneous
?. [UV Crosslinking]
Count cell concentration (either with automated cell counter or hemocytometer) Note: ensure cells are re-suspended well before counting  Note: for cells that do not easily dissociate into single cells, a separate plate of cells can be counted instead (see * above)
?. [UV Crosslinking]
Resuspend in the desired amount for flash freezing
?. [UV Crosslinking]
Aspirate and discard supernatant.
?. [UV Crosslinking]
Centrifuge the 50 ml conical tube at 200g for five minutes at room temperature.
?. [UV Crosslinking]
After frozen (at least thirty seconds), remove from the liquid nitrogen and store at -80°C.
?. [UV Crosslinking]
Aspirate the supernatant and freeze by submerging the epi-tubes completely in liquid nitrogen.
?. [UV Crosslinking]
Spin down at 200g for five minutes at room temperature.
?. [UV Crosslinking]
Transfer desired amount into 1.5 mL Eppendorf Safe-Lock Tubes (or equivalent)
TITLE: dsRNAs treatment with RNase T1 and DNase I
AUTHORS: Vahid Jalali Javaran

[DESCRIPTION]
For dsRNA sequencing by nanopore sequencing, this protocol was used. Before treating samples with RNase T1, you should measure the total concentration of RNAs in the samples by using a nanodrop or Qubit device, as RNase T1 has the ability to partially digest double-stranded RNAs in the absence of single-stranded RNA.   

[STEPS]
SECTION: Digestion
2. Add 50 units RNase T1 per 1µg of total RNA and 1 unit DNase I per 2µg of total RNA 
SECTION: Digestion
1. Add 10X DNase Buffer with MgCl2 (final concentration should be 1X). 
SECTION: Digestion
3. Incubate at 37 degrees C for 20 min.
SECTION: Inactivation of enzymes
4. cleanup with phenol/chloroform extraction.
TITLE: protocols.ioでプロトコルを公開する
AUTHORS: Ayu  Nagane Uchikawa, Anita Broellochs

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">protocol.ioでプロトコルを公開する方法についてのプロトコルです。</div></div>

[STEPS]
?. [PUBLISHボタンを見つける]
プロトコルのページ(viewモード)で「PUBLISH」ボタンを見つける:
?. [PUBLISHボタンをクリックする]
プロトコルを公開する準備ができたら、「PUBLISH」ボタンをクリックします。
?. [PUBLISHボタンをクリックする]
新しいウィンドウが開きます。
?. [著者の追加]
このプロトコルに貢献したすべての著者を追加してください。テキストフィールドに著者名（複数の場合はカンマで区切る）を入力して「ADD」をクリックするか、「add myself as an author」をクリックして自分を著者として追加してください。また、各著者の所属を追加してください。
?. [プロトコルのステータス]
公開するプロトコルについて、以下のいずれかを選択します:Working（動作します）In development（開発中です）Other （その他）また、選択した内容について説明を加えてください。
?. [プロトコルの種類]
あなたのプロトコルが Research protocol（研究用プロトコル）かNon-research protocol（非研究用プロトコル）かを選択してください。
?. [確認]
Confirm that your co-authors and supervisor agree to the publication and that you understand that the protocol will be published and permanently available by checking the boxes: 共同執筆者と監修者が公開に同意していることと、プロトコルが公開され永久に利用可能であることをあなたが理解していることを、ボックスにチェックを入れて確認します。
?. [出版]
最後に「PUBLISH」をクリックすると、プロトコルが protocols.ioで公開されます。
TITLE: U Michigan - Optokinetic Measurements of Visual Acuity and Contrast Sensitivity
AUTHORS: David A. Antonetti

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary: </span></div><div class = "text-block">A virtual optometry system is used to quantify the spatial vision of laboratory animal.</div></div>

[STEPS]
?. Animal is placed inside a square box displaying a rotating cylinder comprised of a vertical sine wave grating is calculated and drawn in virtual three-dimensional coordinate space on four computer monitors facing the animal to form a square.
?. Animal stands unrestrained on a platform in the center of the square
?. The animal’s head movement is tracked for reflexive head and neck movements in response to the grating rotating around the animal
?. The spatial frequency of the grating is clamped at the viewing position by repeatedly re-centering the cylinder on the head in real time
?. Visual acuity is quantified by increasing the spatial frequency of the grating until an optomotor response could not be elicited
?. Contrast sensitivity is measured by identifying the minimum contrast that generates tracking over a range of spatial frequencies
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p><span style="color: #000339; font-family: Raleway, sans-serif; font-size: 14px; font-style: normal; font-variant-ligatures: normal; font-variant-caps: normal; letter-spacing: normal; orphans: 2; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; widows: 2; word-spacing: 0px; -webkit-text-stroke-width: 0px; background-color: #ffffff; float: none; display: inline !important;">This protocol is part of the manuscript: </span><span style="color: #000339; font-family: Raleway, sans-serif; font-size: 14px; font-style: normal; font-variant-ligatures: normal; font-variant-caps: normal; letter-spacing: normal; orphans: 2; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; widows: 2; word-spacing: 0px; -webkit-text-stroke-width: 0px; background-color: #ffffff; float: none; display: inline !important;"><a href="http://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.2000862" target="_blank">Gonçalves et al. Commensal bacteria and essential amino acids control food choice behavior and reproduction. Plos Biology. 2017 Apr 18.</a></span><strong><span style="color: #000339; font-family: Raleway, sans-serif; font-size: 14px; font-style: normal; font-variant-ligatures: normal; font-variant-caps: normal; letter-spacing: normal; orphans: 2; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; widows: 2; word-spacing: 0px; -webkit-text-stroke-width: 0px; background-color: #ffffff; float: none; display: inline !important;"><br /></span></strong></p>

[BEFORE_START]
<p>Prepare the required fly and bacterial media.</p>

[GUIDELINES]
<p><img src="https://s3.amazonaws.com/pr-journal/iu3fw26.png" data-src="https://s3.amazonaws.com/pr-journal/iu3fw26.png" data-ofn="Screen Shot 2017-03-18 at 1.55.25 AM.png" /></p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;">Prepare yeast-based medium (YBM) as follows:</p>
<ul style="text-align: justify;">
<li>mix 8 g agar, 80 g barley malt syrup, 22 g sugar beet syrup, 80 g corn flour, 10 g soya flour, 18 g instant yeast, 8 ml propionic acid, and 12 ml nipagin (15% in 96% ethanol) and fill up to 1000 ml using milliQ filtered water</li>
<li>autoclave before pouring into polypropylene fly culture vials (VWR, #734-2261)</li>
<li>after solidifying, the food is supplemented with instant yeast granules on the surface</li>
</ul>
<p> </p>
<p>Perform all fly rearing, maintenance, and behavioral testing at 25°C in climate-controlled chambers at 70% relative humidity in a 12-hr-light-dark cycle (Aralab, FitoClima 60000EH).</p>

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

[DESCRIPTION]
<p>This protocol is provided for optimization of miRNA mimics transfection in a 6-well plate. Lipofectamine® RNAiMAX Transfection Reagent is a proprietary formulation for transfecting small RNAs (e.g., siRNA, Silencer® Select siRNA, Stealth® RNAi, mirVana™ miRNA mimics and inhibitors) into a wide range of eukaryotic cells.</p>

[GUIDELINES]
<p>RNA-Lipofectamine® RNAiMAX complexes must be prepared in serum-free medium such as Opti-MEM® Reduced Serum Medium and can be added directly to cells in culture medium, in the presence or absence of serum and antibiotics. It is not necessary to remove complexes or change/add medium after transfection unless toxicity by Lipofectamine® RNAiMAX is observed.</p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: Improved methods for high yield genomic DNA of cat stools using DNeasy PowerSoil Pro Kit.
AUTHORS: Hajar Fauzan Ahmad

[STEPS]
?. Spin the PowerBead Pro Tube briefly to ensure that the beads have settled at the bottom. Add up to 250 mg (ideally 200 to 250 mg) of cat stool and 800 µl of Solution CD1, together with 25 µL of Proteinase K. Vortex briefly to mix.
?. Vortex at maximum speed for 10 min. Upon completion, transfer to Eppendorf ThermoMixer to continue incubating, and mixing almost for 20 minutes at 65°C.
?. Centrifuge the PowerBead Pro Tube at 15,000 x g for 1 min. Transfer the supernatant to a clean 2 ml Microcentrifuge Tube (provided).
?. Add 200 µl of Solution CD2 and vortex for 5 s. Centrifuge at 15,000 x g for 1 min. Avoiding the pellet, transfer up to 700 µl of supernatant to a clean 2 ml Microcentrifuge Tube (provided).
?. Add 600 µl of Solution CD3 and vortex for 5 s.
?. Load 650 µl of lysate to an MB Spin Column. Centrifuge at 15,000 x g for 1 min. Discard the flow-through and repeat step 8 to ensure that all of the lysate has passed through the MB Spin Column.
?. Carefully place the MB Spin Column into a clean 2 ml Collection Tube (provided). Avoid splashing any flow-through onto the MB Spin Column.
?. Add 500 µl of Solution EA to the MB Spin Column. Centrifuge at 15,000 x g for 1 min. Discard the flow-through and place the MB Spin Column back into the same 2 ml Collection Tube.
?. Add 500 µl of Solution C5 to the MB Spin Column. Centrifuge at 15,000 x g for 1 min.
?. Discard the flow-through and place the MB Spin Column into a new 2 ml Collection Tube (provided). Centrifuge at up to 16,000 x g for 2 min. Carefully place the MB Spin Column into a new 1.5 ml Elution Tube (provided).
?. Add 100 µl of Solution C6 to the center of the white filter membrane, and let the C6 remain at filter membrane for 10 minutes.
?. Centrifuge at 15,000 x g for 1 min. Discard the MB Spin Column. The DNA is now ready for downstream applications.
TITLE: Effectiveness of rehabilitation for osteoarthritis of the knee associated with isolated meniscus injury: a scoping review protocol
AUTHORS: Masateru Hayashi, Shusaku Koga, Takashi Kitagawa

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Objective: The purpose of this scoping review is to examine whether there are differences in rehabilitation according to the degree of damage to the isolated medial and lateral meniscus and to determine whether it is of value to conduct a systematic review in the future.</div><div class = "text-block">⮚ Introduction: The prevalence of meniscus injuries in middle-aged and elderly people (>50 years) is 31%, and they occur more frequently in medial meniscus injuries than in lateral meniscus injuries (28%＞12%). The treatment of meniscus injuries consists of conservative therapy and surgery, and the benefit of combining surgery with rehabilitation has become clear. However, the effectiveness of rehabilitation alone, according to the degree of isolated meniscus injury, has not been clarified.</div><div class = "text-block">⮚ Eligibility criteria: The review will be a scoping review of the effectiveness of rehabilitation for osteoarthritis of the knee with unilateral or bilateral isolated meniscus injuries in patients over 40 years of age. Outcomes will be pain, function, and re-injury. All types of research will be accepted without restrictions as to location, race, gender, or language of the original article. ⮚ Methods: A systematic search of PubMed, Cumulative Index to Nursing and Allied Health Literature (CINAHL), Web of Science, and Physiotherapy Evidence Database (PEDro) databases using the keywords "meniscus" and "physical therapy" will be conducted during September 2021. In addition, we will not limit the language of the search, in order to include all studies.</div><div class = "text-block">Data extraction tools will be used to specifically show findings in extracted articles related to participants, concepts, context, research design, and review questions. Outcomes should be pain, function, and re-injury, and results should be specified for each outcome.</div></div>

[STEPS]
TITLE: Detoxil 600mg Kapseln Avis France 2020- Avantage or Medical Forum Arnaque
AUTHORS: health  

[DESCRIPTION]
L'utilisation régulière des pilules doit obtenir les résultats souhaités nécessaires. La seule chose que l'utilisateur doit garder à l'esprit est que sauter des repas ne l'aidera pas à perdre du poids, il devra suivre un régime alimentaire normal pendant l'utilisation du produit.

[STEPS]
1. Detoxil  : Vous sentez-vous gêné en regardant votre silhouette dans le miroir ? Tu sais ce que je veux dire? Eh bien, cela fait très mal parce que vous faites de votre mieux pour vous mettre en forme même en faisant des heures régulières et en faisant de l'exercice, mais vous êtes déçu maintenant et je vous dis pourquoi ? Parce que vous ne recevez pas de supplément dans votre alimentation ou si vous n'avez pas reçu le bon supplément qui fonctionne vraiment pour votre corps, alors maintenant, dans cette page Web, nous allons parler des quelques choses dont vous devez vous souvenir lors du choix du supplément également quel supplément vous conviendra le mieux pour vous mettre en forme facilement. C'est l'un des meilleurs compléments sur le marché de nos jours, car il est basé sur un régime cétogène qui correspond exactement à ce que vous voulez et ce ton complet de votre corps dans l'état de cétose où vous brûlerez facilement votre graisse sans aucun effet secondaire. supplément de perte qui est censé perdre votre poids rapidement et c'est un produit assez bien sur le marché ainsi que dans la demande des consommateurs car c'est une formule à action rapide que chaque utilisateur aime si vous attendez exactement que les suppléments ne devraient pas attendre à déplacer et cliquez simplement sur le bouton de commande pour le personnaliser. Detoxil C'est trop tôt, rassurez-vous ! Continuer la lecture. C'est l'un des meilleurs compléments de nos jours car il peut aider votre corps à brûler votre graisse en énergie au lieu des glucides qui ressemblent exactement à un chat qui serait utilisé si l'utilisateur n'a pas pu taper pendant de longs mois est l'un des le meilleur supplément de ce jour est parce qu'il peut aider votre corps à brûler votre graisse en énergie au lieu de glucides, ce qui ressemble exactement à un chat qui serait utilisé si l'utilisateur n'a pas pu taper longtemps, 
Site officiel : https://ipsnews.net/business/2022/07/03/detoxil-avis-france-detoxil-600mg-en-pharmercie-prix-medical-composition-perte-de-poids-gelules-acheter-en-france/
2.
TITLE: DNT Induction
AUTHORS: Zhujun Wei

[STEPS]
?. The DNT powder (>99%) was dissolved in 80% acetonitrile solution, prepared as 10g/L stock solution, and stored at 4℃.
?. The glycerin strains were first grown overnight in LB medium at 37℃ supplied with appropriate antibiotics.
?. The bacteria cultured overnight were diluted by 1/100 times into fresh LB medium, and a certain amount of DNT stock was added to make DNT reach an expected concentration. Then, the samples (200 µL) were added to the wells of a clear 96-well plate with LB medium only as a negative control.
?. The RFU (relative fluorescence units) and OD600 were measured every 30 minutes using the multi-functional enzyme readerat 37°C shaking culture condition, for a period of 12h. (For RFU measurement, 200 µL of LB was used for calibration, with an excitation wavelength 485 nm, an excitation bandwidth 20 nm, an emission wavelength 535 nm, and an emission width 70 nm.)
?. The experiment was repeated three times for each sample.
TITLE: Single cell passaging of hPSC 
AUTHORS: Valeria Fernandez Vallone, Lyn Healy, Nathalie Lefort, Katarzyna Ludwik, Tamer Onder, Lisa Pavinato, Fatma Visal FVO OKUR, Harald Stachelscheid

[DESCRIPTION]
This protocol describes single cell passaging of hPSC using enzymatic dissociation reagents. 

[GUIDELINES]
The protocol is adaptable to different enzyme-free reagents described in Materials section and various vessel formats.

[STEPS]
SECTION: hPSC single-cell passaging
9. Transfer hPSC suspension to a 15 or 50 mL conical tube.
 

SECTION: hPSC single-cell passaging
10. Centrifuge at 300 x g for 5 min.
SECTION: hPSC single-cell passaging
5. Add enzymatic dissociation reagent, refer to Table 1 for recommended working volumes. 
SECTION: hPSC single-cell passaging
7. Add hPSC media supplemented with survival factors to the wells containing dissociation reagent, refer to Table 1 for recommended harvesting volumes. 
SECTION: hPSC single-cell passaging
6. Incubate the vessel at 37 °C for 5 min -  10 min. 
 

SECTION: hPSC single-cell passaging
8. Pipette up and down several times to dislodge the cells from the culture surface. 
 

SECTION: hPSC single-cell passaging
11. Aspirate and discard supernatant without disrupting the cell pellet.
SECTION: hPSC single-cell passaging
12. Resuspend cell pellet in hPSC media supplemented with survival factors. Resuspension volume will depend on pellet size in a range of 1-10 mL.
SECTION: hPSC single-cell passaging
13. Perform hPSC counting using viability staining such as Trypan Blue 0.4%.
 

SECTION: hPSC single-cell passaging
14. Adjust hPSC suspension volume according to desired number of cells for seeding, refer to Table 1 for recommended final media volumes. 
 

SECTION: hPSC single-cell passaging
15. Aspirate coating solution from the destination vessel.
SECTION: hPSC single-cell passaging
16. Pipette cell suspension into the coated recipient wells according to desired hPSC seeding density, refer to Table 1 for recommended final media volumes. 
SECTION: hPSC single-cell passaging
17. Gently move destination vessels with hPSC in suspension in cross shape to distribute them evenly.
SECTION: hPSC single-cell passaging
18. Incubate the vessels at 37 °C  and at 5 % volume CO2.
SECTION: hPSC single-cell passaging
19. After 1440 min, perform full media change with respective hPSC maintenance media without survival factors.  For further hPSC culture refer to protocol Maintenance of hPSC.
 


SECTION: Preparation of destination vessel
1. Prepare coated tissue culture vessels and culture media according to hPSC line requirements and desired format (Table 1).
Refer to protocols: Coating of tissue Culture Vessels for hPSC and Maintenance of hPSC. 
 
 
SECTION: Preparation of destination vessel
2. Equilibrate prepared destination vessel at 37 °C until usage.
SECTION: hPSC single-cell passaging
4. Aspirate and discard medium from the selected source vessel.
SECTION: hPSC single-cell passaging
3. Prepare required volume of the reagents and media according to the Table 1.  Equilibrate the media to Room temperature.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p><a target="_blank"></a>Extracted DNA is quantified using the Quantus fluorometer (Promega) with QuantiFluor®</p>
<p>Double stranded DNA (dsDNA) system according to the instructions in the user manual. The QuantiFluor® dsDNA System contains a fluorescent DNA binding dye which enables sensitive and specific quantification of small amounts of dsDNA.</p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: Immunohistochemistry Protocol
AUTHORS: Hong-Yuan Chu

[DESCRIPTION]
This protocol describes immunohistochemistry.

[STEPS]
SECTION: Preparation
1. Make up 1 L of fresh Phosphate Buffered Saline (PBS) from 10x stock solution.

SECTION: Preparation
2. Make up PBS-T: 0.2–0.5% Triton X-100 in PBS (stir 100-250 mg Triton in 50 mL of PBS).

SECTION: Primary reactions
3. Place each series in a glass vial and rinse mouse brain sections with PBS 3 times before starting primary reactions.
SECTION: Primary reactions
4. Add 1 mL of PBS-T with 2% Normal Donkey Serum (20 µL) to each series and swirl briefly.

SECTION: Primary reactions
5. Optional blocking step: leave slices in PBS-T and 2% Normal Donkey Serum at Room temperature for 45–60 min.

SECTION: Primary reactions
6. Add primary antibody to each series.
SECTION: Primary reactions
7. Shake gently for 2880 min at 4 °C (sections should barely revolve around the vial).

SECTION: Secondary reactions
8. Rinse sections with PBS 3 times before starting secondary reactions.
SECTION: Secondary reactions
9. Add 1 mL of PBS-T with 2% Normal Donkey Serum (20 µL) to each series and swirl briefly.

SECTION: Secondary reactions
10. Add secondary antibody to each series.
SECTION: Secondary reactions
11. Shake gently for 90 min at Room temperature, protected from light (sections should barely revolve around the vial).
SECTION: Secondary reactions
12. Rinse sections with PBS 3 times before mounting.
SECTION: Secondary reactions
13. Mount sections serially on slides with Prolong Diamond Anti-fade mounting media; protect slides from light and keep at 4 °C after 1440 min drying at Room temperature.
TITLE: Keeping beetles alive in transport with wood-flour media
AUTHORS: Allan Gonzalez, Jiri Hulcr

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes how to safely transport bark and ambrosia beetles. </div><div class = "text-block"><span>This protocol is part of the Bark Beetle Mycobiome (BBM) Research Coordination Network. For more information on the BBM international network: Hulcr J, Barnes I, De Beer ZW, Duong TA, Gazis R, Johnson AJ, Jusino MA, Kasson MT, Li Y, Lynch S, Mayers C, Musvuugwa T, Roets F, Seltmann KC, Six D, Vanderpool D, & Villari C. 2020. Bark beetle mycobiome: collaboratively defined research priorities on a widespread insect-fungus symbiosis. Symbiosis 81: 101–113 </span><a href="https://doi.org/10.1007/s13199-020-00686-9" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://doi.org/10.1007/s13199-020-00686-9</span></a><span>.</span></div></div>

[STEPS]