eamag/protocols-with-steps huggingface dataset:
TITLE: Bacterial abundance from grass litter - Flow cytometry
AUTHORS: cweihe 

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is used to get the abundance of bacteria via flow cytometry from ground, decomposing grass leaf litter. </div></div>

[STEPS]
?. Add  of Pi-buffered GTA solution to ground grass litter sample in a 50 ml tube.
5000 µl
0.1 g
?. store fixed sample in the dark at  for up to 30 days
4 °C
?. When you are ready to run your samples on the flow cytometer add  of 0.1M TSP buffer  and vortex.
550 µl
?. Put samples from step 3 in ultrasonic bath  at for 30 min.
4 °C
?. Prepare vacuum manifold (Hoefer FH225) by placing 15 ml tubes on the bottom of manifold in a holder. Make sure filtration nozzles of the filtration unit lead into the 15 ml tubes. If you have more than 10 samples to filter keep the remaining samples at   in the dark until they can be filtered. Add 25 mm 2.7 um filter on the filter support grids and then place the chimney weights on top. Make sure the tube from the pump is connected with the vacuum connector. Turn on vacuum pump and pour samples from step 4 into the chimney weight and open the teflon valves. Wait until all liquid is filtered through. After the filtration remove chimney weights carefully, remove top part of the manifold and close all the 15 ml tubes. Take out the 15 ml tube holder from the filtrate collection box. Replace the top on the filtrate collection box and rinse the filtration support grid with DI water. Then rinse with ethanol let vacuum run for a while to dry. In the mean time clean chimney weights with soap and water and dry them. Then clean the chimney weights with ethanol. Re-assemble for more filtrations.
4 °C
?. Take the 15 ml tubes from step 5 vortex sample and transfer  into a dark microcentrifuge tube. From there prepare a 1:10 dilution with a total volume of  into a new tube with 1% Pi-buffered GTA.
600 µl
600 µl
?. Stain the 1:10 diluted sample with 3 ul of 200 x  nucleic acid stain and vortex. Final concentration of Sybr green should be 1x. Incubate at   for 15 min.
0 Room temperature
?. On the Novocyte Flow cytometer use green fluorescence channel (FL1) and forward scatter (FSC-H) detectors to reduce the natural autofluorescence found in environmental samples. Vortex each sample before running on the Flow cytometer. Each sample should be run at 40 ul / min for 1 min. Make sure that the events/sec stay between 100-1500 events/sec to get more reliable counts. Samples might need to be diluted differently if this number gets too high. Run unstained Pi-buffered GTA as a control for autoflourescence of the buffer. And stained Pi-buffered GTA as a control that the buffer was not contaminated. Run a few unstained samples to account for autoflouresence from environmental conditions.
?. Take absolute counts (evnets/ul) from the gate and subtract the absolute counts within the gate from the background sample. Multiply this by the dilution factor and finally by 1000 to get counts/ml.
TITLE: TAP media preparation
AUTHORS: Joao Vitor Molino

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol describes the preparation of TAP media. Usually used for growing algae cells, as Chlamydomonas reinhardtii. The protocol is derived from the protocol descrived at </span><a href="https://www.chlamycollection.org/methods/media-recipes/tap-and-tris-minimal" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://www.chlamycollection.org/methods/media-recipes/tap-and-tris-minimal/</span></a><span>. </span></div><div class = "text-block">[Gorman, D.S., and R.P. Levine (1965) Proc. Natl. Acad. Sci. USA 54, 1665-1669]</div></div>

[STEPS]
?. [Components mixing]
Add approximatelly   of ddH20 to a large enough vessel. Add a magnetic bar and start and keep mixing with a magnetic stirrer during the preparation. Place pH probe electrode in the solution for pH monitoringFor   Add   Add    Add   Add   Add   , then add drops until   is reachedStop mixing, and add dd H20 until   is reachedStart mixing again until complete mixing is achieved. (5 minutes should suffice).For Add Add Add Add Add  , then add drops until  is reachedStop mixing, and add dd H20 until  is reachedComponets concentration and informations.   ABCDE1Stock solutionComponentAmount for Stock (g) *Check final volume for each in column AMolecular weight (g/mol)Final media concentration (mM)2Solution A (1000ml)NH4Cl4053.497.483MgSO4 . 7H2010246.470.4064CaCl2 . 2H2O5147.010.3456Phosphate solutionK2HPO427174.20.6207(250mL)KH2PO414136.0860.4189Tris Solution (1000mL)Tris242.28121.14201011Acetic acidAcetic Acid Glacial60.05~17.5Hutner`s trace composition and protocol for preparation can be found here.
[ final media volume]
[2M Tris base   (e.g. Trizma) ]
[Solution A]
[Phosphate solution]
[Hutner`s trace solution]
[Glacial acetic acid]
[final volume]
ABCDE1Stock solutionComponentAmount for Stock (g) *Check final volume for each in column AMolecular weight (g/mol)Final media concentration (mM)2Solution A (1000ml)NH4Cl4053.497.483MgSO4 . 7H2010246.470.4064CaCl2 . 2H2O5147.010.3456Phosphate solutionK2HPO427174.20.6207(250mL)KH2PO414136.0860.4189Tris Solution (1000mL)Tris242.28121.14201011Acetic acidAcetic Acid Glacial60.05~17.5
?. [Liquid transfer]
Typically: 1L flasks with blue capsErlenmeyers with a max volume capacity of 100 mL are filled with 50 mL, capped with alluminium foil (2 layers)Erlenmeyers with a max volume capacity of 500 mL are filled with 250 mL, capped with alluminium foil (2 layers)
?. [Autoclavation]
Place flasks inside the autoclave (For flasks with lid, make sure it is loosen enough to allow vapor passage)Set autoclavation to   for at least   , 15 psi.After autoclavation, wait media to cool down and it is ready for use.Set autoclavation to  for at least  , 15 psi.
121 °C
TITLE: Schistosoma mansoni cercariae transformation (without needle)
AUTHORS: Sarah K Buddenborg

[DESCRIPTION]
Free-living aquatic S. mansoni cercariae transform into the first intramammalian stage, called schistosomula or somules, by burrowing in the host skin. Upon contact, cercariae begin to enter the skin and lose their tails, becoming schistosomula. Somules migrate through the epidermis to the dermis to find a small venule or lymphatic vessel to enter the vasculature.  

Transformation of cercariae to schistosomula can be mimicked in the laboratory by centrifuging cercariae to remove tails and then culturing the somules in somule media. This method is particularly useful when the number of cercariae is low (i.e. clonal cercariae from an individual snail).

Somules can be cultured for several weeks with regular media changes.

[BEFORE_START]
Place 1 sterile 15ml falcon tube on ice per snail to be shed
Pre-chill benchtop centrifuge to 4°C
Prepare 1x DPBS+2% anti-anti and place in 37°C reciprocating water bath (see recipes in "MATERIALS" section)
Prepare somule media and place in 37°C reciprocating water bath (see recipes in "MATERIALS" section)

[GUIDELINES]
Media changes and opening of transformed somules to take place in tissue culture hood using sterile techniques

[STEPS]
SECTION: Cercariae collection
1. Shed cercariae from snails in a 6-well plate (see protocol "Schistosoma mansoni cercariae shedding"). The snails can be shed for up to 2 hrs by collecting cercariae and replacing with fresh water every 30 min
2. Using a sterile transfer pipette, dispense cercariae into sterile 15ml falcon tubes on ice
3. After collecting cercariae, adjust the volume in each 15ml falcon tube to 15ml with sterile water
SECTION: Cercariae tail removal
5. Centrifuge the cercariae 2200 rpm, 30 min, 4 °C
6. Quickly remove the supernatant and resuspend the pellet in 10ml pre-warmed 1x PBS + 2% PSF (see "MATERIALS" section for recipes) by gently inverting the tube (do not use pipet to mix)
7. Centrifuge the cercariae  1500 rpm, 10 min, 4 °C 
8. Repeat steps 6 and 7 six more times

SECTION: Somules in culture
9. Resuspend pellet of somules in ~5ml pre-warmed 37 °C somule media (see "MATERIALS" section for recipes)

4. Incubate tubes containing cercariae for 30 minon ice 

SECTION: Somules in culture
11. Incubate at 37 °C, 5% CO230 min

SECTION: Somules in culture
12. Optional: the following day, remove tails with transfer pipet (the tails will have floated to the top of the water column in the wells)  

SECTION: Somules in culture
10. 
Place somules in a 6-well plate and top up each well with pre-warmed 37 °C somule media so each has a total of approximately 4ml media
TITLE: Induced Neurons for the Study of Neurodegenerative and Neurodevelopmental Disorders
AUTHORS: Evelyn J. Sauter, Lisa K. Kutsche, Simon D. Klapper, Volker Busskamp

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Patient-derived or genomically modified human induced pluripotent stem cells (iPSCs) offer the opportunity to study neurodevelopmental and neurodegenerative disorders. Overexpression of certain neurogenic transcription factors (TFs) in iPSCs can induce efficient differentiation into homogeneous populations of the disease-relevant neuronal cell types. Here we provide protocols for genomic manipulations of iPSCs by CRISPR/Cas9. We also introduce two methods, based on lentiviral delivery and the piggyBac transposon system, to stably integrate neurogenic TFs into human iPSCs. Furthermore, we describe the TF-mediated neuronal differentiation and maturation in combination with astrocyte cocultures.</div></div>

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>A chronic illness in childhood has a negative impact on the paediatric patient and on family functioning. Psychological stress in parents influences the level of adjustment to the illness of their children. The Pediatric Inventory for Parents (PIP) was designed to measure stress in parents whose child has a chronic illness or requires prolonged medical monitoring. The main objective of this study is to provide a brief version of the Spanish version of the PIP, across a sample consisted of 465 main familial carers (85.2% female, n=396) between 27 and 67 years old ( =44.13; SD=5.35), of paediatric patients between 9 and 18 years old ( =12.10, SD=2.20;  56.8% men, n=264) diagnosed with diabetes mellitus type I (20.9% of the sample; n=97), short stature (32.5% of the sample; n=151) or a chronic respiratory disease (asthma, cystic fibrosis, bronchiolitis obliterans and bronchiectasis) (46.6% of the sample; n=217). After performing several EFAs (Exploratory Factor Analyses) and CFAs (Confirmatory Factorial Analyses), it was decided that 30 items need to be removed. Reliability and validity results of the new 12-item version suggest appropriate psychometric properties. Cronbach’s alpha value ranging between α=.42 and α=.81 and fit values obtained indicate a good fit: χ2/df (88.393/48) = 1.84 (α &lt; .01); S-B χ²(df)= 88.393 (48); CFI=.95; IFI=.95; RMSEA=.05 (.033 - .074) for the frequency scales and χ2/df (72.002/48) = 1.5 (α &lt; .01); S-Bχ²(df)= 72.002 (48); CFI=.97; IFI=.97; RMSEA=.04 (.011 - .063) for the difficulty scales. The PIP also showed predictive ability about anxiety and depression, a positive relationship between the instrument's own scales and a negative relationship with the caregiver's age. Finally, Differences in stress levels were found depending on the paediatric patient's diagnosis. In order to facilitate the interpretation of the data, centiles were calculated for the whole sample and for each pathology separately.</p>

[STEPS]
?. 
?. 
?. 
?.
TITLE: Preparing feeder-free hPSCs for nucleofection
AUTHORS: Hanqin  Li, Oriol Busquets, Steven Poser, Dirk Hockemeyer, Frank Soldner

[DESCRIPTION]
This protocol describes the standard procedure preparing feeder-free human pluripotent stem cells (hPSCs) for nucleofection. 

General notes
1. Throughout this protocol, the term hPSC is used to collectively refer to both hiPSCs and hESCs. All described procedures have been tested and work equally well for hiPSCs and hESCs.
2. This protocol is to prepare cells for protocol nucleofection of hPSCs. Before starting, familiarize yourself with the protocol and the required preparations. A detailed protocol on maintaining MEF-cultured hPSCs can be found in the collection "Maintenance and inactivation of mouse embryonic fibroblasts (MEFs) as feeder cells for human pluripotent stem cell culture;" doi:
3. Detailed protocols for preparing plasmids, RNA, and RNP for nucleofection can be found in the collection "Nucleofection (Amaxa) and electroporation (Biorad) of hPSCs." A link to this collection can be found in the title section of this protocol, located above.



[STEPS]
1. Cells are ready for nucleofection when the culture reaches 70-80% confluency

For a detailed protocol on growing feeder-free hPSCs, refer to the collection "Feeder-free culturing of hPSCs;" dx.doi.org/10.17504/protocols.io.b4mcqu2w
2. Coat 6-well plates with VTN/Matrigel/Geltrex as depicted in the collection “Feeder-free culturing of hPSCs," dx.doi.org/10.17504/protocols.io.b4mcqu2w
3. Wash hPSCs with DPBS 
4. Use 1 ml Accutase/well of a 6-well plate.
5. Incubate  5 min 37 °C 

6. Add 2 ml DMEM/F12 to each well.
7. Collect all cells into 15 ml conical tube.
8. Add 7 ml DMEM/F12.
9. Centrifuge at 200-300 x g, 5 min 

10. Aspirate supernatant 
11. Resuspend cell pellet in 1 ml DMEM/F12, triturate to single cells using P1000 tips
12. Take two sets of 10 µl of cell suspension. Mix each set with 10 µl trypan blue dye, which comes with the Countess™ Cell Counting Chamber Slides
13. Count cells with Countess automated cell counter or hemocytometer, average the counts from the two sets. Continue with re-suspending the cell pellet in 20 ml nucleofection solution as described in the protocol "Nucleofection of hPSCs" (Step 2)

The protocol "Nucleofection of hPSCs" can be found in the collection "Nucleofection (Amaxa) and electroporation (Biorad) of hPSCs." A link to this collection can be found in the title section of this protocol, located above.


14. Mix the cell suspension in the conical tube, take 500,000 cells per nucleofection reaction and transfer to a new conical tube
15. Centrifuge at 200-300 x g, 5 min 

16. Aspirate supernatant
17. Re-suspend cell pellet in 10 ml DPBS 
18. Centrifuge at 200-300 x g, 5 min 

19. Aspirate supernatant as much as possible, to minimize the interference to the nucleofection buffer system.
TITLE: Restriction Digest -- CHEM 584
AUTHORS: New England Biolabs, Ken Christensen

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The following is a "typical" restriction endonuclease reaction. Please see the "guidelines" tab below for the NEB tips on optimizing restriction digests.</div></div>

[STEPS]
?. Set up the following reaction (total reaction volume 50µl).  AB1Restriction Enzyme10 units is sufficient, generally 1µl is used2DNA1 µg310X CutSmart Buffer5 µl (1X)4Total Reaction Volume50 µl5Incubation Time1 hour*6Incubation TemperatureEnzyme dependent* Can be decreased to 5-15 minutes by using a Time-Saver™ Qualified enzyme.
AB1Restriction Enzyme10 units is sufficient, generally 1µl is used2DNA1 µg310X CutSmart Buffer5 µl (1X)4Total Reaction Volume50 µl5Incubation Time1 hour*6Incubation TemperatureEnzyme dependent
Enzyme volume should not exceed 10% of the total reaction volume to prevent star activity due to excess glycerol.
A 50 µl reaction volume is recommended for digestion of 1 µg of substrate.
The enzyme should be the last component added to reaction
Keep Enzyme in the enzyme storage box while at the bench rather than removing it and placing it on ice.
?. Mix components by pipetting the reaction mixture up and down, or by "flicking" the reaction tube. Do not vortex the reaction.
?. Quick ("touch") spin-down in a microcentrifuge.
?. Incubate for 1 hour at the enzyme-specific appropriate temperature.
Can be decreased to 5-15 minutes by using a Time-Saver™ Qualified enzyme.
See the NEB Activity/Performance Chart for the incubation temperatures.
TITLE: PROTOCOL for “Systemic inflammation triggers long-lasting neuroinflammation and accelerates neurodegeneration in a rat model of Parkinson’s disease overexpressing human α-synuclein”
AUTHORS: mariangela.massarocenere, Valerio Chiurchiù, Nicola Biagio Mercuri

[DESCRIPTION]
Methodological collection for characterization of a dual-hit animal model to assess Parkinson's disease-like symptoms progression 

[STEPS]
TITLE: OMS Atlas FFPE Spatial Mapping
AUTHORS: Brett    Johnson, Danielle  Galipeau, George  Thomas

[DESCRIPTION]
This protocol describes the procedure by which the OMS Atlas serially sections a FFPE block, prepares the resulting slides, and then distributes the specimens for downstream analysis.

[BEFORE_START]
Transfer FFPE blocks to OHSU Knight Histopathology Shared Resource (HSR) for sectioning and processing. Coordinate execution of this sectioning protocol to allow for shipping within the same business week.

[STEPS]
SECTION: Preparation
1. Verify the identity of the FFPE block to be cut against written request for sectioning.
SECTION: Preparation
2. Label all slides with a unique BEMS ID and slide number, corresponding to the written request and FFPE spatial map (below).
 

SECTION: Sectioning
3.  Align block on microtome to minimize tissue loss.
SECTION: Sectioning
4. Face into block at 5μm until full section of tissue is achieved.
SECTION: Sectioning
5. Cut adequate ribbon at 5μm to cover all 8 serial sections.
SECTION: Sectioning
6.  Mount tissue sections onto Tanner slides to maintain serial order and orientation of sections.
SECTION: Processing
7.  Perform hematoxylin and eosin (H&E) staining on slides #1 and #8.
8. Bake slides #2 through #7.

8.1. Place slides #2 through #7 in 55 °C oven overnight.
8.2.  The next morning, raise the oven temperature to 65 °C for up to 60 min.
Note: Slides should be baked at 65 °C for at least 30 minutes.

8.3.  Remove slides from oven. Place slides in slide boxes and store at room temperature.
9. Deliver slides #2 through #7 to BioLibrary for distribution.
TITLE: How to Improve the Reliability of qPCR Detection with Target Gene-optimized Internal Standards
AUTHORS: Jonathan Phillips, Gregor Blaha

[DESCRIPTION]
Achieving robust qPCR results requires both sensitive and accurate detection of target genes. This is particularly challenging for pathogen detection in field samples because the absence of a detection signal does not necessarily indicate the absence of the pathogen. Inhibitors present in the sample can suppress the pathogen signal, leading to false-negative results. The presented protocol details the use of an optimized internal standard for detecting CLas in HLB-infected citrus samples.

[STEPS]
TITLE: Shipping live Daphnia
AUTHORS: Molly Fredericks, Jeff Dudycha, Carla Caceres, Matt Bruner

[DESCRIPTION]
Background:                                                                                                                                                                   
Daphnia are small zooplankton often used in laboratories as model organisms due their well-studied
ecology and evolutionary history, small body size, short generation time, parthenogenetic reproduction, and simplicity of culturing under laboratory conditions. Occasionally laboratories may need to send different species or genotypes to collaborators for various reasons. 

Purpose:                                                                                                                                                                                     
The purpose of this protocol is to set forth a recommended way in which to ship Daphnia specimens from one
laboratory to another in a way that minimizes loss. 

[GUIDELINES]
We have established that this protocol works for multiple species, including D. pulex, D. pulicaria, D. dentifera, D. magna, D. ambigua, D obtusa, and D. lumholtzi.  It should work more broadly.

[STEPS]
1. Label 50mL conical/falcon tubes with the species, genotype, and date in which the Daphnia were placed into the tubes.
2. Fill up the 50mL conical/falcon tubes with 45ml of filtered lake water or Artificial Daphnia Media (ADaM). Other Daphnia media, such as COMBO, may work.
3. Place up to 6 individuals per tube.
4. Feed individuals about 0.5mL of dilute Ankistrodesmus falcatus green algae or similar, then seal with cap.
5. Use parafilm to further secure the cap and tube by placing parafilm around the cap several times.
6. Place the tubes into a Ziploc bag or Whirlpak for secondary containment.
7. Use a box that is size appropriate for however many samples are being sent. Within the box make sure there is an insulated cooler box to help regulate temperature.
8. Place 1-3 frozen icepacks into the bottom of the cooler box and place the bags containing the tubes of Daphnia on top of the icepacks.
9. Double check to make sure nothing is leaking, if there is a leak grab a new tube and repeat the above steps.
10. 10.   Add packaging material such as bubble wrap to secure the tubes and prevent as much movement as possible during shipping.
11. 11.   Close and secure the package with packing tape and either procure stickers that say live animal or write on the box live animal, can also include on the box the species.  In some cases, it may be sufficient to describe the contents simply as "water samples."
12. 12.   Print appropriate shipping label with sender and recipient information.
13. 13.   It is highly recommended to send these samples overnight for next day delivery.
14. 14.   Use a parcel service such as UPS or FedEx to ship to the appropriate destination.   UPS is recommended as their live animal policy includes all crustaceans. FedEx claims that they will accept live crustaceans however depending on the location of shipment processing they may not accept the package.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p><span style="color: #000000; font-family: 'Open Sans', sans-serif; font-size: 13px; font-style: normal; font-variant: normal; font-weight: normal; letter-spacing: normal; line-height: 16px; orphans: auto; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; widows: 1; word-spacing: 0px; -webkit-text-stroke-width: 0px; display: inline !important; float: none; background-color: #ffffff;">This is a collection of G-Biosciences </span><span style="color: #000000; font-family: 'Open Sans', sans-serif; font-size: 13px; font-style: normal; font-variant: normal; font-weight: normal; letter-spacing: normal; line-height: 16px; orphans: auto; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; widows: 1; word-spacing: 0px; -webkit-text-stroke-width: 0px; display: inline !important; float: none; background-color: #ffffff;">FOCUS™ SubCell protocols f</span><span style="color: #000000; font-family: 'Open Sans', sans-serif; font-size: 13px; font-style: normal; font-variant: normal; font-weight: normal; letter-spacing: normal; line-height: 16px; orphans: auto; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; widows: 1; word-spacing: 0px; -webkit-text-stroke-width: 0px; display: inline !important; float: none; background-color: #ffffff;">or the enrichment of subcellular fractions. Please refer to the appropriate protocol depending on your application.</span></p>

[STEPS]
?.