eamag/protocols-with-steps huggingface dataset:
TITLE: Simultaneous detection of miRNA and mRNA at the single-cell level in plant tissues (v2)
AUTHORS: Chi-Chih Wu

[DESCRIPTION]
Detecting the simultaneous presence of a microRNA (miRNA) and a mRNA in a specific tissue can provide support for the prediction that the miRNA regulates the mRNA. Ｗe develop a method that uses sequence-specific miRNA-locked nucleic acid (LNA) and mRNA-LNA probes. Moreover, it augments the detection signal by rolling circle amplification, achieving a high signal-noise ratio at the single-cell level. Dot signals are counted for determining the expression levels of mRNA and miRNA molecules in specific cells. We show a high sequence specificity of our miRNA-LNA probe, revealing that it can discriminate single-base mismatches. Numerical quantification by our method is tested in transgenic rice lines with different gene expression levels. 





[STEPS]
1. Section permeabilization
 
      1. The slides with   sections are taken out the freezer and    equilibrated to RT for 40 mins.    2. Permeabilized in 20 ug/ml proteinase k   for proper duration    3. Quickly wash in DEPC-PBS    4. Quickly dehydrate the slides in EtOH   (50, 70, 99 %) and then air dry     5. Mount the secure seal reaction   chambers onto the slides.    6. Add 1x DEPC-PBS-tween 0.05 % (Wash   buffer) into the chambers to keep the slides wet until RT reaction ready  
 
3. Mixture for mRNA cDNA synthesis
 
        Reagent  Stock  Final    NEB Tag DNA ligase  40U/ul  0.5 U/ul    Rnase H  5 U/ul  0.4 U/ul    Ribolock Rnase inhibitor  40 U/ul  1 U/ul    NEB Tag ligase buffer  10x  1x    BSA  20 ug/ul  0.2 ug/ul    KCl  1 M  0.05 M    Formamide  100%  20%    Pd_A  10 uM  0.1 uM    Pd_B  10 uM  0.1 uM    Pd_C  10 uM  0.1 uM      DEPC-H2O        
 
4. Mixture for miRNA and mRNA padlock probe hybridization and ligation
 
        Reagent  Stock  Final    NEB Tag DNA ligase  40U/ul  0.5 U/ul    Rnase H  5 U/ul  0.4 U/ul    Ribolock Rnase inhibitor  40 U/ul  1 U/ul    NEB Tag ligase buffer  10x  1x    BSA  20 ug/ul  0.2 ug/ul    KCl  1 M  0.05 M    Formamide  100%  20%    Pd_A  10 uM  0.1 uM    Pd_B  10 uM  0.1 uM    Pd_C  10 uM  0.1 uM      DEPC-H2O        
 
5. Rolling circle amplification
 
        Reagent  Stock  Final    Phi 29 polymerase  10 U/ul  1 U/ul    Ribolock Rnase inhibitor  40 U/ul  1 U/ul    10 X phi 29 buffer  10 x  1 x    dNTP  10 mM  0.25 mM    BSA  20 ug/ul  0.2 ug/ul    Glycerol  50%  5%      DEPC-H2O        
 
6. Mixture for detection oligo hybridization
 
        Reagent  Stock  Final    Hyb mixture  4 x  2 x    L13535-S3-FITC  1 uM  0.1 uM    X01178-1852-Cy3  1 uM  0.1 uM    L11805-1854-Cy5  1 uM  0.1 uM      DEPC-H2O        
 
2. Mixture for miRNA hybridization
 
           Stock  Final    Formamide  100%  50%    SSC  20x%  5x    tRNA  10 mg/ml  0.5 ug/ul    Denhardt's  50x  1x    LNA probe A  10 uM  2-3 pmole    DEPC-H2O
TITLE: Making normal NGM for imaging plates (Cabreiro Lab)
AUTHORS: Saul Moore

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">C. elegans is maintained in the laboratory on Nematode Growth Medium (NGM) agar which has been aseptically poured into petri plates. The NGM agar medium can be poured into petri plates easily and aseptically using a peristaltic pump. This pump can be adjusted so that a constant amount of NGM agar is dispensed into each petri plate. A constant amount of agar in the plates reduces the need for refocusing the microscope when you switch from one plate to another.</div><div class = "text-block">The imaging plates can be 35mm, 60mm or 90mm in diameter depending on the assay design.</div></div>

[STEPS]
?. [Pre-Autoclave:]
Book the autoclave (notebook on top of the machine). Take clean flasks from the glass kitchen. Measure all the pre-autoclave reagents and add to the flask (Use a new weighing boat and spatula for each reagent. Also, the cholesterol is kept in the fridge.) Once water is added mix thoroughly and label with autoclave tape (‘NGM Rm 5009’). Make sure the bottle is not screwed completely when placing it inside the autoclave machine.
?. [Using the autoclave:]
- Turn ON the autoclave     - Make sure that the autoclave’s probe bottle is the same size as the largest bottle you use and fill it with water.      - Place the temperature probe in it.     - Fill up the autoclave with water until it reaches the grill.     - Place the bottles in the autoclave and make sure that the cap is not screwed completely.     - Check the waste flask is not too full     - Use ‘media’ program.      - Press START.     - It will take about 2 hours for a 500ml bottle to autoclave and about 2.5 hours for 1L or larger bottles.
?. [Post autoclave:]
When autoclave is complete, remove the probe flask  Make sure to wear gloves as the flask will be hot Let the agar to cool to around 55°C, ie the bottle is cool enough to hold for a second with a gloved hand. Add the post autoclave reagents. Mix it well and start pouring onto imaging plates following the Protocol for Plate Pouring.Try not to shake the bottle too much while mixing to avoid air bubbles.The agar needs to be warm to be poured without blocking the tubings, so try to pour as quickly as possible and if not poured immediately put the bottle on a waterbath set to 60C until being used.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<div title="Page 1">
<div>
<div>
<p>Endo F2 cleaves Asparagine-linked high mannose or biantennary oligosaccharides. It cleaves between the two N-acetylglucosamine residues in the diacetylchitobiose core of the oligosaccharide, generating a truncated sugar molecule with one N-acetylglucosamine residue remaining on the asparagine. In contrast, PNGase F removes the oligosaccharide intact.</p>
</div>
</div>
</div>

[STEPS]
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TITLE: Analysing cellular ATP levels
AUTHORS: Louise Uoselis

[DESCRIPTION]
Protocol for analysis of cellular ATP levels using the Promega Mitochondrial Toxglo Kit

[STEPS]
SECTION: Day 1
1. Seed cells into white opaque walled 96 well plates in 80 µL of media/well, aiming for a confluency of ~80-90% the next day at the time of treatment. Fill all surrounding wells with water to prevent evaporation of the experimental samples. Make sure you seed a DMSO treatment control for each sample collection point. 

SECTION: Day 2
2. Treat cells as desired in 80 µL of media/well

SECTION: Day 2
3. If analysing cells cultured in galactose media, change the media to galactose media 12 – 24 h prior to sample collection. 
SECTION: Day 2
4. To analyse ATP levels, warm the 2x ATP detection reagent to Room temperature, add 80 µL of the 2x ATP detection reagent to each well using a multichannel pipette

SECTION: Day 2
5. Place the plate on a shaker block set to Room temperature, cover the plate in foil, and incubate the plate shaking at 400 rpm for5 min

SECTION: Day 2
6. Immediately take the plate to a plate reader that can read luminescence signal
TITLE: OPestTL V1.4 -- the Open Pesticide Transition Library
AUTHORS: Benjamin Orsburn

[DESCRIPTION]
Pesticide residue screening is a critical method for environmental and food safety. Today, disjointed libraries of pesticide transitions can make it challenging to develop new methods between instrument vendors. From the combined experience of thousands of  separate pesticide screening assays performed by this author, I have compiled a library of over 4,000 pesticide transitions for various instruments. These transitions are presented here in the form of the Open Pesticide Transition Library (OPestTL) along with optimized transitions and settings for insturments from 5 separate instrument vendors. Exact masses are also provided for screening with high resolution instruments in the form of the 3 most common adducts for these compounds. While a growing work, PestTL represents the single largest open library of pesticide transitions assembled to date. v104 contains corrected declustering potentials for relevant instruments and minor corrections caught during the peer review process. 

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<div>
<div>
<div>
<div>
<p>This protocol was designed and developed at this laboratory.</p>
<p>The assay specifically targets the 3' UTR region of DENV-4 strains and is designed as a qualitative screening test for human cases of DENV-4 infection, but not for infection due to other known DENVs.</p>
</div>
</div>
</div>
</div>

[BEFORE_START]
<div>
<div>
<div>
<div>
<div>
<ul>
<li>If using a different brand or model of real-time thermocycler, check the concentration of ROX is adequate.</li>
<li>Method assumes the user is familiar with the thermocycler and software used to run the protocol and with PCR in general.</li>
</ul>
</div>
</div>
</div>
</div>
</div>

[STEPS]
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TITLE: No Title
AUTHORS: 

[GUIDELINES]
<p><strong><u>Materials</u>:</strong></p>
<p>1.)     NC64A mid-log phase cells, 1-2 X 10<sup>7</sup> cells/ml</p>
<p>2.)     PBCV-1 or other virus</p>
<p>3.)     liquid nitrogen</p>
<p>4.)     spatulas, baked</p>
<p>5.)     2 ml microfuge tubes, sterile, RNAse-free (use gloves w/unopened bag, autoclave)</p>
<p>6.)     0.25-0.30 mm glass beads, 250 mg each sample, baked</p>
<p>7.)     chloroform (w/o isoamyl alcohol)</p>
<p>8.)     isopropanol (use a bottle exclusively for RNA work)</p>
<p>9.)     75 % EtOH (use EtOH set aside for RNA work only, dilute w/ DEPC-treated water)</p>
<p>10.)   10X MOPS buffer = 400 mM MOPS, 100 mM NaOAc, pH 7.0 (DEPC-treated)</p>
<p>                  <u>10X MOPS:</u></p>
<p>                  92.48 g MOPS</p>
<p>                  13.61 g NaOAc×3H<sub>2</sub>0</p>
<p>                  Adjust to pH 7.0 w/ NaOH</p>
<p>                  dd-H<sub>2</sub>0 to 1 L</p>
<p>11.)   1 M MgCl<sub>2</sub> solution (DEPC-treated)</p>
<p>12.)   DNAse I (RNAse-free)</p>
<p>13.)   dd-H<sub>2</sub>0 (DEPC-treated)</p>
<p>14.)   1:1 phenol/chloroform (prepared w/o additives for RNA work exclusively)</p>
<p> </p>
<p>Note: DEPC-treat solutions by addition of 0.1% diethylpyrocarbonate, dissolving DEPC by vigorous mixing, incubating 1 h at 37°C or overnight at 25°C, and autoclaving.  Caution: DEPC is toxic.<br /><br />The RNA isolation procedure shown is a modification of the TRIZOL procedure, which is based on a procedure originally developed by Chomczynski and Sachi [Anal. Biochem. <strong>162</strong>, 156-159 (1987)].</p>

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
This protocol is for whole protein extraction from tissues like kidney, liver ,musscle etc. Histone proteins which are tightly twisted on genomic DNA can also be extracted with this protocol.

[BEFORE_START]
ULTRA-TURRAX Dispenser T10 basis S25 (IKA)<br />Rotating mixer<br />Bench top centrifuge

[STEPS]
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TITLE: Enzymatic treatment of free floating sections
AUTHORS: Bryan Killinger

[DESCRIPTION]
General protocol for enzymatic treatment of free floating brain sections.

[STEPS]
SECTION: Prepare 40 micron thick free floating brain sections for enzymatic treatments.
1. Wash in DM
SECTION: Prepare 40 micron thick free floating brain sections for enzymatic treatments.
2. Wash in DM
SECTION: Prepare 40 micron thick free floating brain sections for enzymatic treatments.
3. Wash in DM
SECTION: Equilibrate tissue sections in reaction buffer.
4. For GluC treatment, incubate in 1X GluC reaction buffer (New England Biolabs)
SECTION: Equilibrate tissue sections in reaction buffer.
5. For Trypsin treatment, incubate in TBS (50mM Tris-HCl, pH 7.6, 150mM NaCl)
SECTION: Enzymatic Treatment
6. For Gluc, combine 100ul 2X GluC reaction buffer and 100ul GluC (New England BioLabs, 100 ng/µl dissolved in high-purity water). Incubate tissues with mixture at 37C for 16h.
SECTION: Enzymatic Treatment
7. For Trypsin, combine 4ul of trypsin stock (Sigma-Aldrich, 1 mg/ml dissolved in 1 mM HCl, pH 3) and 196ul of TBS. Incubate tissues with the mixture at 37C for 16h.
SECTION: Wash
8. wash tissue in DM
SECTION: Wash
9. wash tissue in DM
SECTION: Wash
10. wash tissue in DM
TITLE: Aichivirus 3C3D RT-PCR
AUTHORS: Judy Northill

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This RT-PCR will detected Aichivirus A from human samples. It spans the junction region of 3C and 3D and is used for genotyping.</div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/protocols-files/public/a3e3a7e4e0ea7793b0b9d2474be1cf9c08648718f0e1a9a01935b920fe89c7c5/buqghz4w.png" /></div></div>

[STEPS]
?. [Oligonucleotide sequences]
AB1Name 
5'-3'
2AiV-6213FACTGGGCCACCCTCCAGACG3AiV-7044RGGTTGATTTCAGCTTGGAGTTC
AB1Name 
5'-3'
2AiV-6213FACTGGGCCACCCTCCAGACG3AiV-7044RGGTTGATTTCAGCTTGGAGTTC
?. [Reaction set-up]
ABC1Reagent
Vol (µL) x1
Final reaction concentration
2Nuclease free water3.6&nbsp;
3Primer AiV-6213F (20pmol/µl)0.5500nM4Primer AiV-7044R (20pmol/µl)0.5500nM52 X Reaction mix101X6Superscript III RT/Platinum Taq HiFi enzyme mix0.4&nbsp;
7TOTAL VOLUME
15
&nbsp;
Dispense 15µL to each reaction well. Add 5µL of template, extracted RNA, controls or NTC (nuclease-free water).Total reaction volume is 20µL
ABC1Reagent
Vol (µL) x1
Final reaction concentration
2Nuclease free water3.6&nbsp;
3Primer AiV-6213F (20pmol/µl)0.5500nM4Primer AiV-7044R (20pmol/µl)0.5500nM52 X Reaction mix101X6Superscript III RT/Platinum Taq HiFi enzyme mix0.4&nbsp;
7TOTAL VOLUME
15
&nbsp;
?. [Amplification]
The assay has been used with Eppendorf thermocyclers.PCR cycling times  ABCD11 cycle40 cycles1 cycleHold250°C 30 minutes94°C 15 seconds68°C 5 minutes15°C394°C 2 minutes50°C 30 seconds468°C 60 seconds
ABCD11 cycle40 cycles1 cycleHold250°C 30 minutes94°C 15 seconds68°C 5 minutes15°C394°C 2 minutes50°C 30 seconds468°C 60 seconds
?. [Amplification]
Amplified products are analysed by gel electrophoresis or equipment such as a QIAxcel.For gel electrophoresis, a 1.5% agarose gel with ethidium bromide was made using the following recipe.  AB1ReagentVolume2Agarose1.5g30.5 X TBE buffer100mlBoil in microwave for 1.5 -2 minutes until agarose powder is dissolved.Add 2 drops of ethidium bromide (0.625mg/ml) and mix before pouring warm into the gel form with a comb.Leave for approx 30minutes at room temperature to set.Place gel (on a tray) into the tank and cover with 0.5X TBE with ethidium bromide. Mix 2μl of loading dye with10µl of amplified product and add to a well in the gel.Mix 2μl of loading dye with 5μl of 100bp marker and add to a well in the gel. Ideally, the first and last lane but this is not essential.Run the gel for 60-90 minutes at 80 volts or until the bands on the marker have separated adequately without run off the end of the gel.
AB1ReagentVolume2Agarose1.5g30.5 X TBE buffer100ml
For the novice user to gel electrophoresis there a detailed protocol at https://dx.doi.org/10.17504/protocols.io.s38egrw  that will give you further guidance.Experienced users may run their favorite gel recipe with appropriate dye. The aim is to visualise the amplified products. Gel running times and voltage can be adjusted depending on the equipment used.
Amplified products will produce a band of 832bp in size. No temple controls (NTC) should be not detected.
TITLE: iNeuron differentiation from human iPSCs
AUTHORS: Dan Dou, C. Alexander Boecker, Erika L.F. Holzbaur

[DESCRIPTION]
We adapted a previously-described method (Boecker et al., 2020, 2021; Fernandopulle et al., 2018) for differentiating iPSCs stably expressing mNGN2 at a safe-harbor locus into human excitatory glutamatergic neurons. Pre-i 3Neuron iPSCs (human iPSCs with an integrated doxycycline-inducible mNGN2 transgene in the AAVS1 safe-harbor locus) were a gift from M. Ward (National Institutes of Health, Maryland).

[GUIDELINES]
Citations:

Boecker, C.A., and Holzbaur, E.L.F. (2021). Hyperactive LRRK2 kinase impairs the trafficking of axonal autophagosomes. Autophagy 00, 1–3.
Boecker, C.A., Olenick, M.A., Gallagher, E.R., Ward, M.E., and Holzbaur, E.L.F. (2020). ToolBox: Live Imaging of intracellular organelle transport in induced pluripotent stem cell‐derived neurons. Traffic 21, 138–155.
Fernandopulle, M.S., Prestil, R., Grunseich, C., Wang, C., Gan, L., and Ward, M.E. (2018). Transcription Factor-Mediated Differentiation of Human iPSCs into Neurons. Curr. Protoc. Cell Biol. 79, e51.

[STEPS]
SECTION: iNeuron differentiation from human iPSCs
1. Culture pre-iNeuron iPSCs in a 10 cm dish coated with Growth Factor Reduced Matrigel in Essential 8 media, fed daily. 
 

SECTION: iNeuron differentiation from human iPSCs
3. After 1440 min, replace all media with fresh Induction Media, containing 2 μg/ml doxycycline but no ROCK inhibitor. 

SECTION: iNeuron differentiation from human iPSCs
3.1. Replace again with the same media after 1440 min (2880 min after plating).

SECTION: iNeuron differentiation from human iPSCs
4. 72 hours after plating, dissociate cells with warm Accutase. 

SECTION: iNeuron differentiation from human iPSCs
4.1. Count cells in freezing media.


 Freezing media

 

SECTION: iNeuron differentiation from human iPSCs
4.2. Freeze down cells in a Mr. Frosty container placed in a -80 °C freezer  . 

SECTION: iNeuron differentiation from human iPSCs
4.3. On the following day, transfer cryopreserved neurons to liquid nitrogen storage.
SECTION: iNeuron differentiation from human iPSCs
2. Passage iPSCs using warm Accutase and plate 5.5 x 106 cells onto a Matrigelcoated 15 cm dish, in Induction Media (DMEM/F12 supplemented with 1% N2- supplement [GIBCO], 1% NEAA [GIBCO], and 1% GlutaMAX [GIBCO], and containing 2 μg/ml doxycycline and 10 μm ROCK inhibitor).
TITLE: Copy of Copy of          ELISA for quantification of human C6 in serum or plasma.
AUTHORS: Angel  Justiz-Vaillant, Belkis Ferrer-Cosme

[DESCRIPTION]
<div class = "text-blocks"></div>

[STEPS]
?. An anti-human C6 coating antibody is adsorbed onto the microwells by incubation overnight at 4°C with carbonate-bicarbonate buffer.
?. Add 50 µl of human serum or plasma. Human C6 present in the serum or plasma binds to antibodies adsorbed into the microwells.
?. The microplate is blocked with 3% non-fat milk-PBS buffer and later wash to remove unbound proteins.
?. Fifty (50) µl of biotin-conjugated anti-C6 antibody is added. The optimal dilution must be investigated.
?. The microplate is rewashed with PBS-Tween 20 buffer, pH 7.4.
?. One hundred µl of streptavidin-HRP conjugate is added and it binds to the biotin-conjugated anti-C6 antibody. The optimal dilution of this conjugate must be investigated.
?. The plate is washed following incubation to remove the unbound Streptavidin-HRP.
?. Add 100 µl of 3,3',5,5'- tetramethylbenzidine (TMB; Sigma-Aldrich) into each well.
?. Incubate the microwells in the dark for 15 min.
?. A colored product is formed in proportion to the quantity of C6 present in the sample or standard.
?. The reaction is terminated by addition of 100 µl 3M H2SO4  and the absorbance is measured at 450 nm.
?. A standard curve is made from 7 human C6 standard dilutions and the human C6 sample concentration is determined.
TITLE: UC Davis - Hydrogen Peroxide
AUTHORS: Peter Havel

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary:</span></div><div class = "text-block"><span>
Hydrogen peroxide (H</span><span style = "vertical-align:sub;">2</span><span style = "vertical-align:sub;vertical-align:sub;">O</span><span style = "vertical-align:sub;">2</span><span>) is a ubiquitous, toxic, metabolic by-product of aerobic respiration, oxidative stress, and oxidative injury. Cayman's Hydrogen Peroxide Assay Kit utilizes the well established xylenol orange detection method of quantifying the oxidation of ferrous ions (Fe2+) to ferric ions (Fe3+) by hydrogen peroxide. A unique feature of Cayman's assay is the inclusion of catalase as an H</span><span style = "vertical-align:sub;">2</span><span style = "vertical-align:sub;vertical-align:sub;">O</span><span style = "vertical-align:sub;">2</span><span> scavenger for the purpose of confirming the specificity of the reaction for H</span><span style = "vertical-align:sub;">2</span><span style = "vertical-align:sub;vertical-align:sub;">O</span><span style = "vertical-align:sub;">2</span><span>. The sensitivity and the specificity of the assay make it well suited to accurately measure urinary levels H</span><span style = "vertical-align:sub;">2</span><span style = "vertical-align:sub;vertical-align:sub;">O</span><span style = "vertical-align:sub;">2</span><span> in a 96 well plate format. Each kit contains hydrogen peroxide, reagent 1, reagent 2, catalase, a 96 well plate, plate cover, and complete instructions.</span></div></div>

[STEPS]
?. H2O2 Standard Wells - add 20 µl of standard (tubes A-G) and 10 µl of HPLC-grade water per well in the designated wells on the plate (see Sample Plate Format, Figure 1, page 7).
?. Sample Wells - Each sample should have at least two wells that will not contain catalase and two wells that will contain catalase. Add 20 µl of sample to the sample and sample + catalase wells. Then add 10 µl of catalase to the catalase wells and 10 µl of HPLC-grade water to the non-catalase wells
?. Add 200 µl of Working Reagent to each well. Cover the plate with the plate cover and incubate on a shaker for one hour at room temperature.
?. Remove the plate cover and read the absorbance at 595 nm using a plate reader.
?. Calculation1. Calculate the average absorbance of each standard, sample, and sample + catalase.2. Substract the average absorbance of standard A from itself and from all other standards and samples including the catalase containing samples.3. Plot the corrected absorbance of standards (from step 2 above) as a function of the final H2O2 concentration (µM) from Table 1. See Figure 2 (on page 13) for a typical standard curve.4. Subtract the catalase sample absorbance from the non-catalase sample absprbance to yield the corrected sample absorbance.5. Calculate the H2O2 concentration of the samples using the equation obtained from the linear regression of the standard curve substituting corrected absorbance values for each sample.