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

[DESCRIPTION]
<p>This protocol demonstrates how to convert raw long reads produced using a strand-specific sequencing protocol (e.g. ONT's strand-switching protocol) into strand-specific mapped reads.</p>
<p> </p>
<p>The general ides is to use LAST to identify the adapter orientation relative to the genome, and then use that information to reorient called sequences to create a stranded BAM files that is displayable in a genome browser.</p>

[BEFORE_START]
<p>I have written <a href="https://github.com/gringer/bioinfscripts/blob/master/maf_bcsplit.pl" target="_blank" rel="noopener noreferrer">my own script</a> to process LAST results into a CSV format, which makes it easier to do line-by-line data filtering. I have also created a <a href="https://github.com/gringer/bioinfscripts/blob/master/fastx-fetch.pl" target="_blank" rel="noopener noreferrer">fastq filtering script</a> that helps for filtering reads into different files.</p>
<p> </p>
<p>You will also need access to the following free and open-source software programs:</p>
<ul>
<li><a href="http://last.cbrc.jp/" target="_blank" rel="noopener noreferrer">LAST</a></li>
<li><a href="https://github.com/lh3/minimap2#install" target="_blank" rel="noopener noreferrer">minimap2</a></li>
<li><a href="http://www.htslib.org/download/" target="_blank" rel="noopener noreferrer">samtools</a></li>
</ul>
<p>And the following additional data files:</p>
<ul>
<li>a FASTA file containing strand-specific primer / adapter sequences.</li>
<li>a FASTA file containing the genome / sequence of interest.</li>
</ul>

[STEPS]
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TITLE: Immunofluorescence assay (IFA)
AUTHORS: Thanh Ngoc Nguyen

[DESCRIPTION]
This protocol details the procedure of immunofluorescence assay (IFA).

[STEPS]
SECTION: Procedures
1. Seed the HeLa cells onto HistoGrip (ThermoFisher) coated coverslips in growth media in 6 well plates (DMEM supplemented with 10 % (v/v) FBS, 1 % Penicillin-Streptomycin, 25 millimolar (mM) HEPES, GlutaMAX (Life Technologies) and non-essential amino acids (Life Technologies)).
 

SECTION: Procedures
2. Aspirate off the culture media and add 700 µL of fixation buffer to each well and incubate on a side-to-side shaker for 10 min.
SECTION: Procedures
3. Aspirate off the fixation solution and wash three times with 1x PBS.
SECTION: Procedures
4. Permeabilise the cells with Permeabilization buffer (2 mL for each well) for 10 min on a side-to-side shaker.
SECTION: Procedures
5. Aspirate off the Permeabilization buffer and wash once with 1x PBS.
SECTION: Procedures
6. Aspirate off PBS and incubate the cells with Blocking buffer (700 µL for each well) for 15 min on a side-to-side shaker.
SECTION: Procedures
7. Aspirate off the Blocking buffer, wash once with 1x PBS and once with permeabilization buffer.
SECTION: Procedures
8. Aspirate off the permeabilization completely and put the 6 well plates on a flat bench.
SECTION: Procedures
9. Add the primary antibodies (diluted to the right concentrations in Blocking buffer; 15 µL/well) onto the glass coverslips and incubate for 60 min - 120 min depending on the strength of the antibodies.
SECTION: Procedures
10. Wash the coverslips twice with 1x PBS and once with permeabilization buffer.
SECTION: Procedures
11. Aspirate off the permeabilization completely and put the 6 well plates on a flat bench.
SECTION: Procedures
12. Add the relevant secondary antibodies (diluted in Blocking buffer to 1/200-1/500; 15 µL/well) onto the glass coverslips and incubate in the dark for 60 min – 120 min.
SECTION: Procedures
13. Wash three times with 1x PBS and mount the coverslips on microscope slides.
SECTION: Procedures
13.1. Use a yellow tip, drop one small drop off mounting medium onto a microscope slide.
SECTION: Procedures
13.2. Pick out one coverslip from a well (still in 1x PBS) and place it onto a piece of whatman paper so that the side that the cells grew on is facing up.
SECTION: Procedures
13.3. Take the microscope slide with a drop of mounting medium and put it on the coverslip and gently push it down
to remove excessive mounting medium.
SECTION: Procedures
13.4. Seal the edges with nail polish, let it dry and store in a microscope box at 4 °C prior to imaging.
TITLE: Water holding capacity by mass loss
AUTHORS: Jennifer Moore

[DESCRIPTION]
Modified protocol based on that published by Nelson et al., 2024, 55(8):1190-1204.

[STEPS]
SECTION: Set-up
1. Fold a Whatman #1 filter paper into quadrants to make a cone shape.
Place into a conical funnel.
Pre-saturate filter with DI water by dispensing water around the edges of the filter paper and allow excess to pass through.
SECTION: Weighing and Drying
3. Weigh soil plus wet filter paper and record mass to 0.000g. Also, record the mass of the wet, but drained filter (BF0).
Place soil and wet filter paper into an oven set to 105C and allow to dry overnight.
After drying, cool tins for a minimum of 20 minutes. 
After cooling, weigh dry soil plus filter paper and record mass to 0.000g. Also, record the mass of the dried filter (BF0)
SECTION: Wetting up soil
2. Add 8.0g +/- 0.05g of air-dried soil to the wet filter paper. Use top-loading balance. You do not need to record the weight.
Saturate soil by wetting the outer rim of the exposed filter paper so as not to disrupt the soil or cause crusting. A squirt bottle with a narrow tip works best.
After the soil glistens or a small amount of water forms at the surface, stop adding water.
Include one blank filter paper without soil and wet as a control to account for any evaporation (see equation below for more details).
Cover funnels loosely with saran wrap (that you will use for weighing the wet filter paper) to minimize evaporation.
Record time of saturation. Note: CTRL + SHIFT + semi-colon, is the short-cut to add the time in excel.
Allow soils to drain for 6h.
While samples are draining, weigh empty tins and record the tin id # and the mass to 0.000g.
SECTION: Calculation
4.   
MWHC = [SF6 + (BF0 - BF6)] - SFdry/ SFdry

SF6 = mass in g of saturated and drained soil plus filter paper after 6 h drainage
BF0 and BF6 = mass of dry filter and "Blank" filter with no soil but saturated after 6h to account for any water lost via evaporation.
SFdry = mass of oven-dry soil plus oven-dry filter paper in g.
TITLE: Crasher Keto Gummies: Best For Weight Loss| It Trended Buy Now
AUTHORS: DtieSmuh  

[DESCRIPTION]
<!--td {border: 1px solid #ccc;}br {mso-data-placement:same-cell;}-->crasher keto gummies help you increase your energy, and strength, to burn fat fast & get a slim figure *buy now on official site*

[STEPS]
TITLE: SOP16v1_TGD_Qubit_dsDNA_BR_Assays_for_DNAQuantification
AUTHORS: Varsha Rajesh

[DESCRIPTION]
Guidelines
Bench work should be completed on the pre-PCR/DNA bench.
Blue DNA-only labeled materials should be used for this protocol to avoid cross-contamination. 

Lab attire: gloves, lab coat, and safety glasses/goggles.

Before Start
Grab ice.
Use this spreadsheet template to calculate reagent + buffer mix. 
  


[BEFORE_START]
Grab ice. 
Use this spreadsheet template to calculate reagent + buffer mix. 
  

[GUIDELINES]
Bench work should be completed on the pre-PCR/DNA bench.
Blue DNA-only labeled materials should be used for this protocol to avoid cross-contamination. 

Lab attire: gloves, lab coat, and safety glasses/goggles.

[STEPS]
1. Prepare the correct amount of mastermix of Qubit reagent in buffer for your samples and standards. 

Note: A calibration is required for every usage of the Qubit, so always include standards in the calculations. 
Reagent and buffer mastermix is light sensitive. Wrap the tube in foil to protect from light. 
1.1. Reagent must be diluted 1:200 in buffer. 
Each standard requires 190 uL of mastermix.
Each sample requires 190-199 uL of mastermix. (You can put anywhere from 1 to 10 uL of sample in each qubit assay). 

2. Select the reagent calculator on the Qubit Fluorometer main menu. 
Ensure that your calculations match up with the numbers calculated by the Qubit for mastermix preparation.
5. Go back to home screen and select the dsDNA assay: Broad Range. Select "Read Standards".
6. Input Standard 1, then read. Then input Standard 2. Generate calibration curve.
4. Prepare Standards 1 and 2. Each requires 10 uL of standard and 190 uL of mastermix. Keep the standards on ice.
3. Prepare samples: Aliquot the appropriate amount of mastermix (190-199 uL) into each 0.5 mL qubit assay tube. Add the appropriate amount of sample (1-10 uL) into each tube, and let sit for 2 minutes to incubate. 

After incubation, the sample fluorescence is stable for 3 hours. 
7. Enter what sample volume you added to each tube. 
8. After reading one tube, just take out the sample and put in the next tube. This way all samples will be saved under the same assay data set.
9. When finished, upload the data onto a USB.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol describes the complete protocol for performing N-Glycoproteomics from plasma samples. Albumin removal is performed prior to protein digestion with trypsin. Tryptic peptide mixture is used for enriching N-glycopeptides by lectin-affinity chromatography using a lectin-mix. Enriched N-glycopeptides are cleaned up by reverse phase chromatogrraphy and analyzed by LC-MS and LC-MS/MS. resulting MS/MS spectra is deconvouted and saved as peak lits file which can be searched with GlycopeptideId software to identify the peptide sequence, glycan composition and proposed glycan struccture. </p>
<p> </p>

[STEPS]
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TITLE: immunoblotting (JMS protocol)
AUTHORS: Veerle Baekelandt

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">imonoblotting protocol JMS</div></div>

[STEPS]
?. determination of protein content by a bicinchoninic acid (BCA) assay
?. cell extracts containing 15 µg of total protein were separated on 4-15% sodium dodecyl sulfate-polyacrylamide gradient (150V, Bio-Rad) gels
?. electroblot gel onto polyvinylidene difluoride membrane
?. incubate membranes with   paraformaldehyde for  for better protein binding
?. Block membranes with  milk for
?. incubate blot with primary antibody for
[or overnight at 4°C]
?. wash blot for 3 times  for  with PBS- TRITON-X100
?. incubate for  with horseradish peroxidase-conjugated secondary antibody (Dako, Glostrup)
?. detect using chemiluminescence
TITLE: Ligation
AUTHORS: Lizhen Zhu

[DESCRIPTION]
Method of plasmid construction connection (T4 DNA ligase)

[GUIDELINES]
teacher ZHANG 

[STEPS]
SECTION: system
1. 1.5 µL 
4.5 µL 
0.5 µL 
2 µL 
12.5 µL 

SECTION: step1
2. Set up a system as required, all operations on ice Mix ligation system with 100µL of competent and ice bath for at least 30min (thorough contact of DNA and cell, enabling DNA to get inside cell walls)
SECTION: step2
3. 42˚C heat shock for 90s (competent cell swell under high temperature, cell membrane and cell wall fully contact, enabling DNA to flow inside the cell)
Ice bath for 5min (Cell shrinks and DNA is dragged through the cell membrane)
SECTION: step3
4. Pipette 200µL of full nutrient culture (in clean bench), place in a shaker at 37˚C for 1h
SECTION: step3
5. Spread the entire sample on the plate, observe after 16h~20h
6.
TITLE: Video Lecturing in Clicker-assisted English Flipped Class
AUTHORS: zhonggen yu

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Value: The study onthe impact of video lecturing on clicker-assistedEnglishflipped class was necessary because it has seldom been explored.Purpose: This study studied the impact of video lecturing on student satisfaction and English proficiency, plus correlations between student satisfaction levels and English proficiency. Methodology: Randomly recruitedChinese participants (Female N=44; Male N=43)from a university in China received both pre and post College English Test Band 4 and satisfaction measurements, together with a semi-structured interview. Findings: We concluded that the video-assisted class could cause significantly higher English proficiency than the non-video-assistedclass (F=23.17,</span><span style = "font-style:italic;">p<.001</span><span>, Partialη</span><span style = "vertical-align:super;">2</span><span>=.216); there were significant differences between video- and non-video-assisted cohorts for post interaction (F=8.37, p=.005, Partialη</span><span style = "vertical-align:super;">2</span><span>=.093), post efficacy(F=7.68, p =.007, Partialη</span><span style = "vertical-align:super;">2</span><span>=.086), and post regulation (F=16.34, </span><span style = "font-style:italic;">p<.001</span><span>, Partialη</span><span style = "vertical-align:super;">2</span><span>=.166); there were strong, positive relationships between post English proficiency and post student interaction (R</span><span style = "vertical-align:super;">2</span><span>=.70; β=.84; p<.01), self-efficacy (R</span><span style = "vertical-align:super;">2</span><span>=.57; β=.75; p<.01) and self-regulation (R</span><span style = "vertical-align:super;">2</span><span>=.59; β=.77; p<.01) levelsin both cohortsat the </span><span style = "font-style:italic;">.05</span><span> level. However, no strong, positive correlations were found in both cohorts at the</span><span style = "font-style:italic;">.05</span><span> levelbetween pre English proficiency and prestudentinteraction (R</span><span style = "vertical-align:super;">2</span><span>=.00; β=.05; p=.33), self-efficacy [R</span><span style = "vertical-align:super;">2</span><span>=.03;β=-.17 (negative); p=.05] and self-regulation [R</span><span style = "vertical-align:super;">2</span><span>=.05;β=-.23 (negative); p=.01] levels.Future research into video-assisted English flipped class may need interdisciplinary cooperation.</span></div></div>

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Overview of the fragment antigen binding</p>
<p>The fragment antigen binding (Fab fragment) is an antibody structure which still binds to antigens although it is monovalent with no Fc portion. Antibody that is digested by the enzyme papain will yields two fab fragments of about 50 kDa each and an Fc fragment.</p>
<p> </p>
<p>However, fab2 fragment antibodies are produced by pepsin digestion of whole IgG antibodies in order to remove the Fc region while leaving intact some of the hinge region. They have two antigen-binding fab portions linked together by disulfide bionds and then are divalent with a molecular weight of about 110kDa.</p>
<p> </p>
<p>The advantages of fragment secondary <a href="http://www.creativebiolabs.net/Fab-Fragment-Antibodies_24.htm" target="_blank" rel="noopener noreferrer">fab antibodies</a></p>
<ol>
<li>F(ab) and F(ab')2 fragment antibodies eliminate non-specific binding between Fc portions of antibodies and Fc receptors on cells.</li>
<li>Penetrate tissues more efficiently due to their smaller size.</li>
<li>Since fragment antibodies do not have Fc portions, they do not interfere with anti-Fc mediated antibody detection.</li>
</ol>
<p> </p>
<p>Reasons for choosing a <a href="http://www.creativebiolabs.net/Fab-Fragment-Antibodies_24.htm" target="_blank" rel="noopener noreferrer">fab fragment</a></p>
<ol>
<li>Monovalent antibody fragments (F(ab) fragments) are powerful tools to block background from primary antibody binding and in double staining experiments.</li>
<li>F(ab) fragments are used to block endogenous immunoglobulins on cells, tissues and exposed immunoglobulins in multiple labeling experiments using primary antibodies from the same species.</li>
<li>After the blocking step with normal serum, we recommend incubating F(ab) fragments in excess to block endogenous immunoglobulins in IHC. These antibodies are not recommended for blocking immunoglobulins in WB and ELISA.</li>
</ol>
<p> </p>
<p>Reasons for choosing a F(ab')2 fragment</p>
<ol>
<li>Divalent antibody fragments (F(ab')2 fragments) are smaller than whole IgG molecules and enable a better penetration into tissue thus faciliting better antigen recognition in IHC. The use of F(ab')2 fragments also avoids unspecific binding to Fc receptor on live cells or to Protein A/G.</li>
<li>F(ab')2 fragments are not recommended for blocking since they have two binding sites that are available to capture the primary antibody introduced subsequently. However, as opposed to F(ab) fragments, F(ab')2 fragments can both bind and precipitate antigens thanks to their two binding sites. We recommend using normal serum with these antibodies to prevent the binding to Fc receptors.</li>
</ol>

[STEPS]
TITLE: FindingNemo Extraction 4: Circulomics Kit
AUTHORS: Inswasti Cahyani, John Tyson, Nadine Holmes, Josh Quick, Nicholas Loman, Matthew Loose

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This is a sub-protocol designed to extract/isolate ultra-high molecular weight (UHMW) DNA to obtain ultra-long (UL) reads on Nanopore sequencers using the </span><span style = "font-weight:bold;">Circulomics Nanobind CBB Big DNA Kit</span><span>. </span></div><div class = "text-block">A DNA extraction protocol that yields clean and homogeneous UHMW DNA is important for a good UL sequencing output. The choice of protocol should be based on achieving these parameters.</div><br/><div class = "text-block"><span>The </span><span style = "font-weight:bold;">Circulomics Nanobind CBB Big DNA Kit</span><span> was initially the recommended extraction protocol for UL sequencing using SQK-ULK001.</span></div><br/><div class = "text-block"><span>We tested this sub-protocol in </span><span style = "font-weight:bold;">human cell line</span><span>, with standard input cells of 6 millions. As a rule of thumb, a million cells will suffice for one load on a MinION.</span></div><br/></div>

[STEPS]
?. [UHMW DNA Extraction]
Follow the manufacturer’s instructions as described here.
?. [UHMW DNA Extraction]
Store at 4oC or continue to UL Library Preparation as per Section "Modified ULK001". If only SQK-RAD004 is available, follow library preparation as in Section "Modified RAD004".
[for storage]
?. Store at 4oC or continue to UL Library Preparation as per Section "Modified ULK001". If only SQK-RAD004 is available, follow library preparation as in Section "Modified RAD004".
[for storage]
?. [UHMW DNA Extraction]
This protocol is using the Nanobind CBB Big DNA Kit (see Materials).
?. [UHMW DNA Extraction]
Follow the manufacturer’s instructions as described here.
?. [UHMW DNA Extraction]
Store at 4oC or continue to UL Library Preparation as per Section "Modified ULK001". If only SQK-RAD004 is available, follow library preparation as in Section "Modified RAD004" or "KrazyStarFish (KSF)".
[for storage]
?. [UHMW DNA Extraction]
After overnight incubation at room temperature, quantify the DNA as per "UHMW DNA QC" and check homogeneity by calculating %CV values.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>PNGase F cleaves N-linked (asparagine-linked) oligosaccharides from glycoproteins. The enzyme deaminates asparagine to aspartic acid, leaving the oligosaccharides intact. Denaturation increases the rate of cleavage. Most native proteins can still be completely N-deglycosylated but incubation time must be increased. The enzyme will remain fully active under reaction conditions (37˚C) for at least 96 hours. PNGase F will not remove oligosaccharides containing Alpha-(1,3)-linked core fucose commonly found on plant glycoproteins; for this purpose, use peptide N-glycosidase A.</p>
<p> </p>
<p>There are a number of alternative enzymes which can be used to remove N-glycans, most especially the Endo F family of enzymes and Endo H. These enzymes cleave between the two N-acetylglucosamine residues in the core of the oligosaccharide, generating a truncated sugar molecule with one N-acetylglucosamine residue remaining on the asparagine. This leaves a charged sugar which can assist in keeping proteins in solution that precipitate after deglycosylation with PNGase F which removes the oligosaccharide intact. Endo F1 cleaves high mannose and some hybrid type N-glycans. Endo F2 will removes biantennary and high mannose (at a 40X reduced rate). Endo F3 releases of triantennarry and fucosylated biantennary N-glycans. Endo H removes hybrid or high mannose glycans.</p>

[STEPS]
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TITLE: Dual-Choice Chamber Assay
AUTHORS: Natalie L. Kendziorski, Tse-Yu Chen, Sara Farless, Sandra A. Allan, Chelsea T Smartt

[DESCRIPTION]


The protocol describes a laboratory assay to test the repellency of an
essential oil using a modified dual-choice chamber assay system. The assay
assesses the movement of the mosquito away from or towards the essential oil.
The modified system detects repellency. Mosquito responses to repellent
 compounds can be evaluated in a laboratory dual-choice chamber system. 




[STEPS]
1.
TITLE: Quick Protocol for Monarch® PCR & DNA Cleanup Kit (5 ?g) (NEB #T1030)
AUTHORS: New England Biolabs

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This is the "quick" version of Monarch® PCR & DNA Cleanup Kit (5 μg) Protocol (NEB #T1030). For the full protocol, please click </span><a href="https://www.neb.com/protocols/2015/11/23/monarch-pcr-and-dna-cleanup-kit-protocol" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">here</span></a><span>.</span></div></div>

[STEPS]
?. Dilute sample with DNA Cleanup Binding Buffer according to the table below. Mix well by pipetting up and down or flicking the tube. Do not vortex.    ABC1Sample&nbsp;TypeRatio of Binding Buffer: SampleExample2dsDNA &gt; 2 kb&nbsp;(plasmids, gDNA)2:1200 μl: 100 μl3dsDNA &lt; 2 kb (some amplicons, &nbsp;fragments)5:1500 μl: 100 μl4ssDNA&nbsp;&gt; 200 nt*7:1700 μl: 100 μl*Please note that recovery of ssDNA Oligonucleotide Cleanup Protocol, but doing so will shift the cutoff size for DNA binding to 18 nt (versus 50 nt).
ABC1Sample&nbsp;TypeRatio of Binding Buffer: SampleExample2dsDNA &gt; 2 kb&nbsp;(plasmids, gDNA)2:1200 μl: 100 μl3dsDNA &lt; 2 kb (some amplicons, &nbsp;fragments)5:1500 μl: 100 μl4ssDNA&nbsp;&gt; 200 nt*7:1700 μl: 100 μl
A sample volume of 20–100 μl is recommended. For smaller samples, TE can be used to adjust the volume. For diluted samples larger than 800 μl, load a portion of the sample, proceed with step 2, and then repeat as necessary.
?. Insert column into collection tube and load sample onto column. Spin for 1 minute, then discard flow-through.
?. Re-insert column into collection tube. Add 200 μl DNA Wash Buffer and spin for 1 minute.
Discarding flow-through is optional.
?. Repeat Step 3: re-insert column into collection tube. Add 200 μl DNA Wash Buffer and spin for 1 minute.
?. Transfer column to a clean 1.5 ml microfuge tube.
Use care to ensure that the tip of the column does not come into contact with the flow-through. If in doubt, re-spin for 1 minute.
?. Add ≥ 6 μl of DNA Elution Buffer to the center of the matrix. Wait for 1 minute.
?. Spin for 1 minute to elute DNA.
Typical elution volumes are 6–20 μl. Nuclease-free water (pH 7–8.5) can also be used to elute the DNA. Yield may slightly increase if a larger volume of DNA Elution Buffer is used, but the DNA will be less concentrated. For larger size DNA (≥ 10 kb), heating the elution buffer to 50°C prior to use can improve yield.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<div>
<div>
<div>
<div class="b-start-block editable-content">
<p>This protocol is for the whole genome de novo assembly and annotation for apostichopus japonicus genome, but can be useful in other marine invertebrate genomes. It accompanies the following <em>GigaScience</em> publication:</p>
<p> </p>
<p>Jihoon Jo, et al.  (2016): Draft genome of the sea cucumber Apostichopus japonicus and genetic polymorphism among color variants. <em>GigaScience</em>...</p>
</div>
</div>
</div>
</div>

[STEPS]
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