eamag/protocols-with-steps huggingface dataset:
TITLE: Thawing Frozen Adherent Cell Lines
AUTHORS: Carolina Lopez

[DESCRIPTION]
This protocol describes the procedures from thawing and expanding adherent cell lines

[STEPS]
SECTION: Protocol
1. Day 1
Prepare media ahead of time and warm to room temperature
Spray down each bottle with 70% Ethanol before placing it into the TC hood
Pre-cool the centrifuge to 4ºC
Prepare and label a 6-well plate, and aliquot 9 mL of TCM into a 15 mL conical for each tube of cells to be thawed
Find the location of the cells you will be thawing in the liquid nitrogen inventory google doc
Transfer the cells between the liquid nitrogen and the TC room on ice
Record the information on the cryotube – date, number of cells, etc
Begin adding media from the 15mL conical to the still frozen cells, then transfer media back and forth between the two tubes until the cells have completely thawed and everything has been transferred to the 15 mL conical
Spin down the cells at 1200rpm for 10’ at 4ºC 
Pour off the supernatant and break up the cell pellets by tapping the tubes
Add 1 mL of TCM to the cells, pipette up and down to break up any clumps, add 9 mL TCM, and transfer 1-2 mL of resuspended cells to a 6-well plate (cells should reach 80% confluency in two days)
Add 10 µl of Mycoplasma removal agent per 1 mL of media
Confirm the presence of cells in the well by microscope
Incubate at 37ºC
SECTION: Protocol
2. Day 3 *If cells tested negative for mycoplasma before freezing, only 1 round of MRA treatment is necessary. If mycoplasma presence is questionable or unknown 2 rounds of MRA treatment should be done – see mycoplasma protocol for more information.
Warm media to room temperature
Remove media and wash cells once with PBS
Add 1 mL of 0.05% Trypsin-EDTA to wells and incubate plate at 37ºC for 1-3 minutes until cells are completely released from the plate
Add 4 mL of media to wells to halt trypsinization and transfer to a T25 flask
Incubate at 37ºC
SECTION: Protocol
3. Day 4
Warm media to room temperature
Check for cell adherence
If there are a lot of dead cells, change the media on the cells
If cells are nearing 50% confluence, trypsinize, transfer in 10 mL to a T75 flask – Label as passage #0
TITLE: THP-1 differentiation
AUTHORS: Juliane C Fernandes

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Differentiation of THP-1 monocytic cell line in macrophage-like cells</div></div>

[STEPS]
?. [Plating and starting differentiation process]
Centrifuge  at   using
cultivated in RPMI 10% FBS Pen/Strep in a humidified incubator at   5% CO2. Carefully check for any contamination before initiating the protocol.
Centrifuge: 200 34, 8 min
?. [Plating and starting differentiation process]
Estimate the number of bottles necessary to achieve enough cells for the experiment. After the first round of centrifuging, discard culture media and centrifuge the content of new bottles in the same tube.
?. [Plating and starting differentiation process]
Count and evaluate cell viability placing  of the 1:1 cell:Trypan mixture in a Neubauer ChamberCalculate the concentration of cell per mL (Medium of cells/chamber × 2 × 104)
10 µl
?. [Plating and starting differentiation process]
Resuspend pelleted cells in a volume sufficient for counting (usually 5 mL for each centrifuged bottle)
?. [Plating and starting differentiation process]
Mix  of the resuspended cells with  of Trypan Blue.
10 µl
10 µl
?. [Plating and starting differentiation process]
Dilute cells/mL according to the preferred plate for the follow-up experiment24 well plate - 1 × 106 cells/well -  /well (flow cytometry)6 well plate - 5 × 106 cells/well -  /well (RNA extraction)
1 mL
4 mL
?. [Plating and starting differentiation process]
Add PMA.Add PMA from the diluted stock (1 µg/mL). First, dilute 100x to obtain an aliquot of 10 ng/µL. Final concentration for THP-1 differentiation: 30 ng/mL diluted in RPMI 10% FBS.
?. [Plating and starting differentiation process]
Store for  at the incubator
[5% CO2]
?. [Resting]
Aspire cell culture media with a vacuum pump. Wash cells 3 times with PBS 1X to remove residual PMA.
?. [Resting]
Incubate cells with 1 or 4 mL - respectively for 24 well and 6 well plates - of fresh RPMI 10% FBS medium for further
[5% CO2]
?. [THP-1 macrophages]
Proceed to the assay of interest with fully differentiated THP-1 macrophages.
This protocol was tested to the following assays: miRNA inhibition, Leishmania infection, flow cytometry, and RNA extraction.After differentiation cells stop duplicating and adhere to the bottom of plate wells (THP-1 monocytic cell line grow in suspension).
TITLE: Food preference assay of C Elegans
AUTHORS: Priota Islam

[STEPS]
?. [Preparing Bacterial solution of choice (Worm food)]
Fresh bacterial culture of OP50 and HB101 were prepared following the protocol Making OP50 solution from Frozen Stock
?. [Preparing worms]
Maintain N2s on NGM plates seeded with either OP50 or HB101 or a mixture of both depending on experiment planChunk each set of worms on 3 maintenance platesBleach the worms after 2 days following the protocol for Bleach synchronisation of C. elegans  Refeed the starved L1s on 2 different plates that are seeded with either OP50 or HB101 or a mixture of both respectively, 72hr prior to the experiment day
https://www.protocols.io/view/bleach-synchronisation-of-c-elegans-stbeein
?. [Preparing Imaging plates]
Prepare and pour low peptone NGM onto 35mm imaging plates Put them in the cold room for at least 2 days before useOP50 and HB101 were diluted in M9 to the desired dilution factor (1:1, 1:2, 1:4, 1:8 or 1:10)Label and mark the imaging plates according to the experiment plan (Use two dots to mark OP50 and one dot for HB101 making sure they are about 1.5cm apart)Seed imaging plates with two 10ul drops of the diluted bacterial solutions the day before recording (the control plates will either have two drops of OP50 or two drops of HB101, the rest of the plates will have one drop of OP50 and one drop of HB101)Leave on bench to dry with lid on overnight
?. [Day of recording]
Using a hair pick – pick 3 young adults onto the seeded imaging plates  Put the plates on the tracker – agar side up with the lids off  Wait 30min for the worms to acclimatise  Record for 2 hours (See protocol Imaging on the multiworm tracker for detailed tracking instructions)
{"blocks":[{"key":"ejq95","text":"Caenorhabditis elegans, a small (adults are ~1 mm long), free-living soil nematode that feeds on bacteria, is an ideal organism for applying various live microscopy techniques. The organism is transparent, thus it is possible to microscopically analyze and record the whole animal throughout its entire life. Its complete cell lineage is known, making it possible to follow developmental and differentiation processes in real time.","type":"unstyled","depth":0,"inlineStyleRanges":[],"entityRanges":[],"data":{}}],"entityMap":{}}
TITLE: 16S Metagenomic Sequencing Library Preparation (Modified version of Illumina)
AUTHORS: Khine WT Wei

[DESCRIPTION]
Metagenomic studies are commonly performed by analyzing the prokaryotic 16S ribosomal RNA (16S rRNA) gene. This protocol describes a workflow for preparing samples for sequencing the V3-V4 regions of the 16S rRNA gene.

[STEPS]
TITLE: Nerve tissue processing for transmission electron microscopy (TEM)
AUTHORS: Natalia Biscola, Leif Havton

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Transmission electron microscopy (TEM) studies promote an improved understanding of functional studies and provide critical ultrastructural information. This document provide a step by step protocol to embed tissues for TEM analysis. First, the animals are transcardially perfused with 2% paraformaldehyde and 1.25% glutaraldehyde diluted in 0.12M Millonigs buffer (MB), pH 7.3, for 15-20 minutes. After dissection, the tissues are postfixed in the same fixative overnight at 4°C and then rinsed in MB 3 times for 30 minutes each. Embedding protocol includes osmication, dehydration, and embedding of tissues in plastic resin. The tissue blocks are trimmed and semithin sections (0.5 </span><span style = "font-style:italic;">μ</span><span>m) are obtained and stained with 1% toluidine blue solution. Light microscopy image capturing is performed at 100X magnification for detailed analysis. </span></div></div>

[STEPS]
?. [nerve tissue collection]
Animals are transcardially perfused with 0.12M Millonigs buffer (MB), ph=7.3, followed by 2% paraformaldehyde and 1.25% glutaraldehyde diluted in MB for
?. [Dehydration]
Continuing the dehydration place 100% propylene oxide in the tissues for
?. [Fixation]
Fix tissues with 1% Osmium solution diluted in ddH2O for  (Place vials with tissue +Osmium solution in circular shaker)
*Osmium is VERY TOXIC. Work in the fume hood and use appropriate PPE.
Prepare Osmium solution at least one day in advance.
?. [Infiltration]
Infiltration step starts with tissues in 50% propylene oxide+ 50% Epon solution for 2 hours. After, place 100% Epon resin in vials overnight.
Epon Solution:Use Eponate 12 Kit, with DMP-30 (Ted Pella #18010).To prepare 100mL Epon: Mix 47.6mL of RESIN; 32.0mL of DDSA; 23.0mL of NMA; and 1.6mL of DMP-30
?. [Semi-thin sectionning]
Cut semi-thin cross section (0.5μm) and place them on  glass slides.
?. [Semi-thin sectionning]
Label sections with 1% toluidine blue solution diluted in ddH2O for
?. [Semi-thin sectionning]
Wash sections with ddH2O, dry and coverslip with DPX medium.
?. [Image acquisition]
Acquire and analyze images (10X magnification for oveview image and 100X magnification for detailed image) using a Nikon Eclipse E600 microscope and Nikon camera DS-Fi3
?. [nerve tissue collection]
After dissection, the tissues of interest are postfixed in the same fixative overnight at 4°C and then rinsed 3 times with PBS for
[each]
?. [nerve tissue collection]
Wash tissues 3 times with ddH2O for
[each]
?. [Fixation]
Wash tissues 3 times with ddH2O for
[each]
?. [Dehydration]
Dehydrate tissues with alcohol solutions in different concentrations, as follow: 30%, 50%, 70, 80, 95, and 100% (twice) solution diluted in ddH2O
[each]
Use 200 Proof Alcohol to prepare the solutions.
?. [Embedding]
Block tissue of interest in plastic molds. Place the tissue in a direction to obtain cross sections later.
TITLE: PRESTO-Tango Assay
AUTHORS: Abigail  Cornwell

[DESCRIPTION]
PRESTO-Tango Assay protocol for assessing GPR68 activation by benzodiazepines at acidic pH.

[BEFORE_START]
Acidic media preparation (modified from Pera et al.): Rehydrate VWR Traceable pH/ORP meter (10539-802) in pH4.0 solution at least 30 minutes before use. Prepare HTLA media pH6.8 and pH7.4. Follow the instructions for 1L of powdered DMEM (Sigma Aldrich D5030) without adding sodium bicarbonate. When the media is fully dissolved add the following components:  1% Penicillin-Streptomycin (Corning, Mt 30-002-CI), 100ug/mL Hygromycin B (Thermo Scientific, 10687010), 10% FBS (Corning, 10687010), 2ug/mL puromycin dichloride (Thermo Scientific, A1113802) , 1mM sodium pyruvate (Thermo Scientific, 11360070), 12.5mM HEPES (Thermo Scientific, 11360070). Aliquot media into separate beakers and adjust the pH using 10N NaOH/HCl. After adjusting the pH sterile filter the media with a MilliporeSigma™ Stericup™ Quick Release-GV Vacuum Filtration System, 500mL, 0.22um pore size (Fisher Scientific, S2GVU05RE).

[STEPS]
1. Day 1: Plated 400,000 HTLA cells/2mL of media in each well of a 6-well dish by bulk preparing 13mL media + 2.6x10^6 HTLA cells
2. Day 2: Transfect 500ng GPR68-Tango (Addgene #66371) construct in 5 of the 6 wells using Lipofectamine 3000 (L3000008, Thermo Scientific) according to the Manufacturer's Instructions. Perform a no cDNA control transfection in the 6th well.
2.1. Detailed transfection: Prepare DNA and lipid tubes (x5.5). DNA tube: 688uL serum-free DMEM media, 22uL P3000 reagent + 500ng GPR68-Tango cDNA. Lipid tube: 688uL serum-free DMEM media, 22uL lipofectamine. Also, prepare a negative control without GPR68-Tango cDNA (DNA tube (x1.5) =188uL serum-free DMEM + 6uL P3000 reagent, Lipid tube (x1.5)=188uL serum-free DMEM + 6uL Lipofectamine). Vortex the tubes, then add the contents of the DNA tube to the corresponding lipid tube dropwise. Incubate for 20 minutes at room temperature, flicking occasionally to mix. Add 2mL fresh complete media per well to the cells in the 6-well dish. Place in the 37C, 5% CO2 incubator, overnight.
3. Day 3: Cells were split and counted with a BioRad TC-20 automated cell counter. Re-plated 8,000 transfected cells/30uL pH6.8/pH7.4 media per well onto a white flat bottomed TC-treated Corning 384-well plate (bulk prepare the cells for the number of wells required, for example X10= 80,000 cells in 300uL of media). Place cells in the 37C, 0% CO2 incubator.
4. Day 4: A Tecan D300e digital drug dispenser was used to plate the desired drug concentrations using 10mM drug stocks resuspended in DMSO. DMSO concentration was normalized.
5. Day 5: Luminescence levels in each well was measured using Promega Bright-Glo Luciferase Assay System (Catalog #E2610) according to the manufacturer's instructions.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>The method described here is from the PLoS ONE paper entitled "Joint time delay and Doppler passive acoustic 3D tracking, applied to studies of bats in natural habitats".<br />This method detects acoustic events (possible bat calls) and filters them according to their <br />consistency with each other as well as with extracted kinematics of the bat. This allows to filter out false detections, related to echoes and noise, improves the reconstruction of bats trajectories and allows to study bats behavior.</p>
<p> </p>
<p>This method (with another parameters) can be also potentially applied to the extraction of the trajectory of any fast moving objects, in particular, sound-emitting machinery, such as trajectories of cars before and after accident, and many others.</p>
<p> </p>
<p>This protocol (together with bat-related parameters, such as frequency ranges, intervals between calls, etc) is designed to reproduce the method of Joint time delay and Doppler passive acoustic 3D tracking in a case of tracking bats by an array of 4 ultrasonic microphones.</p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: KAPP-Sen TMC: Fixation of Cells and Nuclei for Chromium Fixed RNA Profiling
AUTHORS: Juliana Alcoforado Diniz, Dylan Baker, Jessica Garofalo, Paul Robson

[DESCRIPTION]
Cells are fixated prior to scRNA-seq according to 10X Genomics protocol CG000478.




[STEPS]
SECTION: Fixation of Cells &amp; Nuclei
1. Centrifuge sample at 350 rcf for 5 min at 4°C.
SECTION: Fixation of Cells &amp; Nuclei
2. Remove the supernatant without disturbing the pellet.
SECTION: Fixation of Cells &amp; Nuclei
3. Add 1 ml Fixation Buffer to the sample pellet and pipette mix 5x.
SECTION: Fixation of Cells &amp; Nuclei
4. Incubate for 1 h at room temperature (20°C) or for 16-24 h at 4°C for long term storage.
SECTION: Fixation of Cells &amp; Nuclei
5. Centrifuge at 850 rcf for 5 min at room temperature.
SECTION: Fixation of Cells &amp; Nuclei
6. Remove the supernatant without disturbing the pellet.
SECTION: Fixation of Cells &amp; Nuclei
7. Add 1 ml chilled Quenching Buffer to the sample pellet and pipette mix 5x and keep on ice.
SECTION: Fixation of Cells &amp; Nuclei
8. Determine cell concentration of the fixed sample using AO/PI (acridine orange/propidium iodide) Cell Viability Kit for Luna-FL automated cell counter.
SECTION: Fixation of Cells &amp; Nuclei
9. Thaw Enhancer (10x Genomics PN-2000482) for 10 min at 65°C. Vortex and centrifuge briefly. Keep warm and verify no precipitate before use.
SECTION: Fixation of Cells &amp; Nuclei
10. Add 1 volume pre-warmed Enhancer to fixed sample in Quenching Buffer. Pipette mix.
SECTION: Fixation of Cells &amp; Nuclei
11. Store sample at 4°C for up to 1 week.
SECTION: Fixation of Cells &amp; Nuclei
12. For long term storage (up to 6 months) also add 50% glycerol for a final concentration of 10% with Quenching buffer and store at -80°C.
TITLE: Rearing Bark and Ambrosia Beetles from Naturally Infested Wood
AUTHORS: Jiri Hulcr, Andrew J. Johnson, Demian F Gomez

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes how to rear bark and ambrosia beetles in naturally infested wood. </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]
TITLE: Nuclei isolation from mouse lung for single nucleus RNASeq
AUTHORS: Jeffrey Koenitzer, Ben Humphreys

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol is for</span><span style = "font-weight:bold;"> nuclei isolation from mouse lung for single nucleus RNASeq</span><span style = "font-weight:bold;">.</span></div><div class = "text-block">It is adapted directly from Joshi et al., with adjustments to RNase inhibitor concentrations and removal of FACS sorting steps. </div></div>

[STEPS]
?. Pre-cool all instruments (including centrifuges), buffers, and tubes.
Note: All steps are performed   or in cold room ( ) to minimize RNA degradation.
?. Remove mouse lung sample from -80 ºC freezer and trim a ~6 mm3 piece.
?. Transfer the minced tissue and buffer to a GentleMacs C tube.
?. Run m_lung_01 program and m_lung_02 program in sequence, stopping the latter after .
?. Reduce foam by centrifugation ().
Centrifuge: 750 34, 1 min
?. Wash strainer with .
[Wash Buffer]
?. Pass suspension through a 5 µm strainer into 50 ml conical tube.
?. Centrifuge at .
Centrifuge: 600 34, 5 min, 4 10
?. Resuspend in .
[Resuspension Buffer]
?. Count nuclei by hemocytometer and dilute to desired concentration (e.g. 10,000 per µl).
Note: it is easier to resuspend in lower volumes and dilute than to concentrate nuclei via further centrifugation.
?. Thaw on small plastic weighing boat until able to insert 26G needle to tissue, then inject ice cold  to ‘inflate’ the tissue. Add the remaining Lysis Buffer and chop to the smallest pieces possible with scissors (60 s).
[Lysis Buffer]
?. Close, invert, and transfer directly to MACS Tissue dissociator.
?. Place tube .
on ice
?. Proceed to 10x Chromium.
?. Using a wide bore tip, pipette up and down to recover any pelleted material and pass lysate through 40 µm filter to 50 ml conical tube.
TITLE: Utilisation de l&#39;ordinateur &#39;slaker&#39; pour réaliser des QuantiSlakeTests (QST)
AUTHORS: f.vanwindekens

[DESCRIPTION]
slaking lab draft protocol in french

[STEPS]
1. Version 3.0 !
2. Allumer l’ordinateur



USER: slaker


PASSWORD: *


3. Lancer le programme



Dans le terminal (fênetre blanche ou noire qui s’ouvre automatiquement, ou cliquer sur l’icône Xterm), taper: 
AppSlake
 (on obtient donc 
[slaker@kingkong ~]$ AppSlake
 ) et appuyer sur enter.


Une fenêtre Firefox avec l’application s’ouvre, avec deux onglets, sur les ports 8001 et 8002. Souvent bug au démarrage, donc ouvrir un nouvel onglet avec le port 8002.


4. Eteindre l’ordinateur



Sur le bureau: clic droit –> 
logout
 –> 
Shutdown
, ou bouton de démarrage !


5. En cas de bug



Fermer les onglets Firefox de l’application


Dans le terminal déjà ouvert taper Ctrl-C Ctrl-C (pour arrêter), ou dans un nouveau terminal, puis, dans les deux cas, taper : 
killall R
 ( [slakeman@kingkong ~]$ killall R ) et appuyer sur 
enter



Relancer l’application en tapant: 
AppSlake
 ( [slakeman@kingkong ~]$ AppSlake )  et appuyer sur 
enter



Si le bug persiste, redémarrer le pc en tapant dans le terminal: 
reboot
 ( [slakeman@kingkong ~]$ reboot ) et appuyer sur enter. Retourner au point 3.


Si le bug persiste (encore) : Contacter Frédéric Vanwindekens au 0475 95 21 07 ou au fix 4184. S’il y a internet, lui communiquer l’adresse IP du PC. Elle se trouve sur le bureau à coté du mot 
NETWORK
 (ex: 
10.12.102.24
)


Si les balances montrent des chiffres bizarres : ON/OFF 
> OK (pas l'ordi =;-)
 )


6. Mode opératoire:



Lancer le programme (voir partie “Ordinateur”)


Allumer la balance et, s’il y a un niveau, vérifier qu’elle est à niveau (niveau à bulle)


Mettre de l’eau distilée dans le berlin (1800 ml)


Positioner le panier vide suspendu à la balance dans l’eau


Tarer la balance, panier vide sous eau !


Retirer le berlin


Disposer l’échantillon dans le panier, à sec !


Une fois que le numéro d’échantillon est correctement établit dans l’application cliquer sur “Start the Slaketest”


Lorsque le graphique simple commence à se construire, rapidement replacer le panier avec l’échantillon dans l’eau en faisant attention à ce que le panier ne touche pas les bords


Une fois que le slaketest est terminé (lorsque la pente est relativement nulle, générallement à partir de 1000s), cliquer sur “Stop the Slaketest”. Attendre que le graphique disparaisse (cela peut prendre quelque(s) ittération(s))


Pour limiter le risque de bug, il vaut mieux allumer la balance en avance, et faire des échantillons de test en début de journée et début d’après-midi


7. Thanks

Draft by : @WilliamGysselinck
TITLE: Sensitivity and specificity test of toehold
AUTHORS: Hung Liang Pai, Cheng-Ruei Yang

[STEPS]
?. [Preparation]
Sterilize the bench, and put on a labmat
?. [Preparation]
Thaw the reagents including 1. DNase/ RNase free water (store in -20°C) 2. solution A (store in -80°C) 3. solution B (store in -80°C) 4. RNase inhibitor (store in -20°C) 5.toehold switches with invertase DNA (store in 4°C) 6.miRNA as the trigger (store in 4°C)
on ice
?. [Protocols]
Gently shake and spin down the tube before adding (especially solution A and DNA plasmid)
?. [Protocols]
Add below reagents in to PCR tubes in order:1. ddH2O(till totally )2. solution A3. solution B4. inhibitor5. toehold switches with invertase DNA6. miRNA or (depends on whether its control group or test group)
5 µl
2 µl
1.5 µl
0.2 µl
50 ng
50 ng
0 ng
?. [Protocols]
Centrifuge
Centrifuge: 4000 33
?. [Protocols]
Place the PCR tubes in the dry bath incubator
37 °C
?. [Protocols]
Put the PCR tubes   after 2 hours
on ice
?. [Protocols]
Measure the glucose concentrations of each PCR tubes by glucometer
?. [Protocols]
Put the PCR tubes in the dry bath incubator
55 °C
?. [Protocols]
Measure the glucose concentrations of each PCR tubes by glucometer again