eamag/protocols-with-steps huggingface dataset:
TITLE: Protein Expression Using BL21(DE3) (C2527)
AUTHORS: New  England   Biolabs

[DESCRIPTION]
This protocol explains methods for Protein Expression Using BL21(DE3) Competent E. coli (C2527).

You can find the protocol for a large-scale Protein Expression here.

[GUIDELINES]
BL21(DE3) Genotype:
fhuA2 [lon] ompT gal (λ DE3) [dcm] ∆hsdS
λ DE3 = λ sBamHIo ∆EcoRI-B int::(lacI::PlacUV5::T7 gene1) i21 ∆nin5

NOTES:
1. Caution: This product contains DMSO, a hazardous material. Review the MSDS before handling.

2. Storage and Handling: Competent cells should be stored at -80°C. Storage at -20°C will result in a significant decrease in transformation efficiency. Cells lose efficiency whenever they are warmed above -80°C, even if they do not thaw.

[STEPS]
1. Transform expression plasmid into BL21(DE3). 
2. Plate on antibiotic selection plates.
3. Incubate   at 37 °C. 
4. Resuspend a single colony in 10 mL.
5. Incubate at 37 °C until OD600 reaches 0.4–0.8.
6. Induce with 4 µL or 40 µL of 100 Milimolar (mM) (final concentration of 40 or 400 µM).
7. Induce for 3-5 hours at 37 °C.
8. Check for expression either by Coomassie stained protein gel, Western Blot or activity assay. Check expression in both the total cell extract (soluble + insoluble) and the soluble fraction only. 
 

9. For large scale, inoculate 1 L of liquid medium (with antibiotic) with a freshly grown colony or 10 mL of freshly grown culture.

10. Incubate at 37 °C until OD600 reaches 0.4–0.8.
11. Add 40 Micromolar (µM) or 400 Micromolar (µM) IPTG and express protein using optimal time/temperature determined in a small scale trial.
TITLE: Download Sequences from Eurofins - Edit Filenames for Use in Geneious
AUTHORS: Dakota Betz

[DESCRIPTION]
Our protocols are constantly evolving and old versions will be deleted.
The documents here are not intended to be cited in publications

[GUIDELINES]
This protocol is optimized for Macs for use with the bash terminal.

[STEPS]
1. Download sequences from Eurofins (you should have a link in your email). Once downloaded, unzip the folder.
2. Optional: Search your four letter code (use command F in Finder) in the Eurofins folder and move all of your sequence files to another folder that is easy to navigate to. 

For example, create a new folder called Date_Seqs on your Desktop and paste all of the sequences you found with your four letter code there.
3. Open the bash terminal and navigate to the folder with your sequences in it (use the cd command and the path to the folder). 

If you're following the example, see below. Note that all commands will be written in the text in bold, while items in italics indicate parts of the commands that are specific to you and therefore likely to change from the example (see above; includes file/folder paths, sample names, etc).
 



4. Once you've navigated to the folder with your sequences in it, paste in the following to replace your four-letter code with an underscore: for file in *code*; do mv "$file" "$file//code/_}; done 
Be sure to replace "code" with the four-letter code that is specific to you/your files.
 

5. Output: Check that all of your files have been renamed. In the above example, all instances of "code" have now been replaced with an underscore. You should see the same result. You can now proceed with bringing your .ab1 files into Geneious and assembling them as per usual.
TITLE: Feeder-free culturing of hPSCs
AUTHORS: Hanqin  Li, Oriol Busquets, Steven Poser, Dirk Hockemeyer, Frank Soldner

[DESCRIPTION]
This collection describes the standard procedure of feeder-free culturing of human pluripotent stem cells (hPSCs) using mTeSR-plus or StemFlex 

Collection overview
Coating plates
    A. VTN
    B. Matrigel
    C. Geltrex
Adapting hPSCs cultured on MEFs to feeder-free system
Thawing of feeder-free hPSCs 
Passaging of feeder-free hPSCs
    A. Accutase
    B. ReLeSR
Freezing of feeder-free hPSCs
    A. Accutase
    B. ReLeSR

General Notes
Throughout these protocols, 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.
Until otherwise indicated, feeder-free hPSCs are routinely grown in a humidified cell culture incubator under “low” oxygen conditions. We have successfully maintained hPSCs using either 3% O2 (3% O2, 5% CO2) or 5% O2 (5% O2, 5% CO2) conditions.
We have routinely maintained feeder-free cells in either mTeSR-plus or StemFlex. However, these two media are not interchangeable. Pick one and stick to it.
We have routinely maintained feeder-free hPSC cultures on VTN, Matrigel and Geltrex-coated cell culture plates without observing obvious differences.
We have routinely passaged feeder-free hPSCs using either Accutase (as single cell suspension) or ReLeSR (as cell aggregates) without observing obvious differences. 

[STEPS]
TITLE: mRNA purification with Ambion Dynabeads
AUTHORS: Magdalena Julkowska

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The mRNA isolation that is usually used prior to RNAseq library preparation protocol</div></div>

[STEPS]
?. [Adjust the volume of 2 ug of RNA to 36 uL with distilled water]
?. [Heat to 65°C for 2 minutes to disrupt secondary structures]
?. [Transfer 72 uL of well resuspended Dynabeads to microcentrifuge tube - place on magnet rack for 30 seconds or untill all Dynabeads migrate to the tube well]
?. [Discard the supernatant, remove tube from the magnet rack, and add 100 uL Binding Buffer to calibrate the beads. Put back on the magnet rack and remove the supernatant]
?. [Add 36 uL Binding Buffer to Dynabeads. Optimal hybridization required Binding Buffer added in 1:1 ratio relative to sample volume. ]
?. [Add the total RNA to the Dynabeads / Binding Buffer suspension. Mix thoroughly and rotate on a mixer for 5 minutes at room temperature to allow mRNA to anneal to the oligo dT25 on the beads.]
?. [Place the tube on the magnet rack and wash mRNA-bead complex twice with 200 uL Washing Buffer B. Remove all the supernatant between each washing step with help of the magnet. ]
?. [Elute the samples with 10-20 uL of 10 mM Tris-HCl pH 7.5. Heat to 65°C for 3 minutes and place the tube immediately on the magnet rack.]
?. [Transfer the eluted mRNA to a new RNAse free tube]
TITLE: Characterizing the neighborhood risk environment in multisite clinic-based cohort studies: a practical geocoding and data linkages protocol for protected health information
AUTHORS: Ariann Nassel, Marta G  Wilson-Barthes, Chanelle J. Howe, Sonia Napravnik, Michael J. Mugavero, Deana Agil, Akilah J. Dulin

[DESCRIPTION]
Methods. This protocol demonstrates how to: 


(1) securely geocode patients’ residential addresses in a clinic setting and match geocoded addresses to census tracts using Geographic Information System software (Esri, Redlands, CA); 
(2) ascertain contextual variables of the risk environment from the American Community Survey and ArcGIS Business Analyst (Esri, Redlands, CA); 
(3) use geoidentifiers to link neighborhood risk data to census tracts containing geocoded addresses; and 
(4) assign randomly generated identifiers to census tracts and strip census tracts of their geoidentifiers to maintain patient confidentiality. 


Results. Completion of this protocol generates three neighborhood risk indices (i.e., a Neighborhood Disadvantage Index, a Murder Rate Index, and a Assault Rate Index) for patients’ coded census tract locations. 


Intended Usage. This protocol can be used by research personnel and clinic staff who do not have prior GIS experience to easily create objective indices of the neighborhood risk environment while upholding patient confidentiality. Future studies can adapt this protocol to fit their specific patient populations and analytic objectives. 

[STEPS]
TITLE: Survival Analysis
AUTHORS: Núria Peñuelas, Ariadna Laguna, Miquel Vila

[DESCRIPTION]
Mice Survival Analysis

[STEPS]
SECTION: Survival Analysis
1. Registerall births and follow the cohort of animals throughout their lifespan.
SECTION: Survival Analysis
2. Report all natural deaths in the followed cohort. 
SECTION: Survival Analysis
3. Register all animals used for experimental purposes as censored animals, and do not include them in future follow-ups. It is important that the animals chosen for experimental purposes are selected randomly to avoid potential bias in the survival analysis due to biased censoring.
SECTION: Survival Analysis
4. When all survival data has been collected, either as censoring events or as natural deaths, perform the log-rank test and the Gehan-Breslow-Wilcoxon test to assess whether there are differences in survival between distinct groups.
TITLE: No Title
AUTHORS: 

[BEFORE_START]
7 hour IL-3A virus infected NC64A, pellets frozen at -80°C

[GUIDELINES]
<strong>MATERIALS AND ASSAY CONDITIONS:</strong> <br /><br /> 1) 7 hour IL-3A virus infected NC64A, pellets frozen at -80°C<br /> 2) Buffer A: <br />
<ul>
<li>10 mM Tris-Acetate, pH 8.0</li>
<li>10 mM 2-M</li>
<li>50 µg/mL PMSF</li>
</ul>
3) Buffer B: <br />
<ul>
<li>20 mM Tris-Acetate, pH 8.0</li>
<li>0.5 mM EDTA</li>
<li>7 mM 2-ME,10% Glycerol</li>
</ul>
4) Buffer B, pH 8.5: <br />
<ul>
<li>20 mM Tris-Acetate, pH 8.5</li>
<li>0.5 mM EDTA</li>
<li>7 mM 2-ME</li>
</ul>
5) Storage buffer: <br />
<ul>
<li>20 mM Tris-Acetate, pH 8.0</li>
<li>0.5 mM EDTA</li>
<li>0.1 mM DTT</li>
<li>50 mM KOAc</li>
<li>5 mM MgAc, 50% Glycerol</li>
<li>100 µg/mL BSA (the BSA is added after dialysis into the storage buffer from a 10 mg/mL stock)</li>
</ul>
6) 4 M NaCl<br /> 7) 28% Polyethyleneglycol (PEG) 8000<br /> 8) Assay buffer and conditions:<br />       1X CviJI assay buffer:<br />
<ul>
<li>20 mM GlycylGlycine, pH 8.5 (with KOH)</li>
<li>10 mM MgAc</li>
<li>0.1 mM DTT</li>
<li>50 mM KOAc</li>
<li>100 µg/mL casein (optional)</li>
</ul>
All assays are carried out in 20.0 µL volumes with 1 µg of pUC19 DNA as substrate for 60 to 120 min at 25°C. The assays are electrophoresed on 2.0% agarose gels (100 mL gels with double 20 lane combs) for 1 hour at 100 volts in 1X TPE buffer. Gels are stained with 0.5 µg/mL ethidium bromide for 30 min and photographed on a UV light box.

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
Drosophila melanogaster females' productive system has been an excellent model in studying many developmental events such as stem cell specification and cell migration. The ovaries are composed of a chain of egg chambers starting with the germaruim that buds of stage 1 egg chamber after&nbsp;encapsulated it with follicle cells. Then, stage 1 egg chamber goes under 14 developmental stages. &nbsp;This protocol provides the basic method for both dissecting and immunostaining ovaries. Each step is critical. This protocol is modified from&nbsp;http://www.ncbi.nlm.nih.gov/pubmed/8162854

[STEPS]
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TITLE: High-molecular weight DNA extraction, clean-up and size selection for long-read sequencing
AUTHORS: Ashley Jones, Cynthia Torkel, David Stanley, Jamila Nasim, Justin Borevitz, Benjamin Schwessinger

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Rapid advancements in long-read sequencing technologies have transformed sequencing read lengths from bps to Mbps, which has enabled chromosome-scale genome assemblies. However, read lengths are now becoming limited by the extraction of pure high-molecular weight DNA suitable for long-read sequencing, which is particularly challenging in plants and fungi. To overcome this, we present a protocol collection; high-molecular weight DNA extraction, clean-up and size selection for long-read sequencing. We optimised a gentle magnetic bead based high-molecular weight DNA extraction, which is presented here in detail. The protocol circumvents spin columns and high-centrifugation, to limit DNA fragmentation. The protocol is scalable based on tissue input, which can be used on many species of plants, fungi, reptiles, insects and bacteria. It is also cost effective compared to kit-based protocols and hence applicable at scale at low resource settings. An optional sorbitol wash is listed and is highly recommended for plant and fungal tissues. To further remove any remaining contaminants such as phenols and polysaccharides, optional DNA clean-up and size selection strategies are given. This protocol collection is suitable for all common long-read sequencing platforms, such as technologies offered by PacBio and Nanopore. Using these protocols, sequencing on the Oxford Nanopore MinION can achieve read length N50 values of 30-50 kb, with reads exceeding 200 kb and outputs ranging from 15-30 Gbp. This has been routinely achieved with eucalypts, acacias, rice, themeda, wheat, wheat rusts, various other fungi, geckos, skinks, ticks, ladybird beetles, caterpillars and E. coli.</div></div>

[STEPS]
TITLE: Cyanobacteria Total Lipid Extraction from Cell Pellets
AUTHORS: Robbie Martin, Steven W Wilhelm, Katarina A. Jones, Shawn Campagna

[DESCRIPTION]
This protocol is designed/used for extraction of total cellular lipids from cyanobacteria samples (either lab cultures or field samples) collected via centrifugation for use in lipid analysis and quantification via mass spectrometry. 



Please contact Dr. Steven Wilhelm (wilhelm@utk.edu) or Robbie M. Martin (rmarti49@vols.utk.edu) for additional information regarding this protocol.
 
Modified from Guan, X. L., Riezman, I., Wenk, M. R., & Riezman, H. (2010).  Yeast lipid analysis and quantification by mass spectrometry.  Methods in Enzymology, 470, 369-391.
 

[STEPS]
20. Prepare the three separate solutions needed for this extraction protocol as listed below.
 lipid extraction solvent (step 2) with a 1:1 ratio of butanol and lab purified water (water-saturated butanol, and lab purified water
 
1. Prepare the three separate solutions needed for this extraction protocol as follows:
lipid extraction solvent: a 15 : 15 : 5 : 1 : 0.18 ratio by volume of 95% ethanol, water, diethyl ether, pyridine, and 4.2 N ammonium hydroxide, respectively.
water-saturated butanol:  a 1:1 ratio of butanol and Milli-Q water
purified lab water: (Milli-Q water) 
2. Concentrate cells via centrifugation using methods appropriate for your available lab equipment and for your species of interest.  Decant supernatant.  Transfer pelleted cells to a 2-mL centrifuge tube.  Cells may need to be re-pelleted and residual supernatant removed after transfer to final 2-mL centrifuge tube.

Note: Appropriate volume of lab culture or field samples to concentrate and extract depends on cell concentration.  As a guideline, we have been successful concentrating 10-25 mL of lab cultures of Microcystis aeruginosa and ~50 mL of either raw lake water or mesocosm samples.
3. Add 1 mL of extraction solvent, ~100 µL of glass beads, and vortex ~5 s. 
4. Incubate sample in 60 °C water bath for 20 min.
5. Centrifuge sample at 10,000 x g for 10 min.
6. Remove supernatant and place into a 1-dram glass vial (dram vial #1).  The first two extractions from a sample will be placed in this vial (#1).
7. Repeat steps 3-6, except DO NOT ADD more glass beads.
8. Dry the collected supernatant in dram vial #1 under a stream of nitrogen.
9. Re-suspend dried sample in 300 µL of water-saturated butanol and 150 µL of Milli-Q water.
10. Vortex and transfer to a 2-mL centrifuge tube.
11. Centrifuge at 10,000 x g for 2 min. 
12. Remove top butanol phase and place into a NEW 1-dram glass vial (dram vial #2).
13. Wash original dram vial (#1) with 300 µL saturated butanol and transfer to residual aqueous phase in 2-mL centrifuge tube from step 10.  Vortex.
14. Centrifuge at 10,000 x g for 2 min.  Remove top butanol phase and place into dram vial #2.
15. Dry the collected butanol phase in dram vial #2 under a stream of nitrogen.
16. Re-suspend dried sample in 300 µL of 9:1 methanol:chloroform.
17. The samples are now ready for analysis via LC/MS.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol is provided by BioNano Genomics and was used in:</p>
<p> </p>
<p>Huang Zhihai, Xu Jiang, Xiao Shuiming, Liao Baosheng, Gao Yuan, Zhai Chaochao, Qiu Xiaohui, Xu Wen, Chen Shilin</p>
<p> (2016): Supporting data for 'Comparative optical genome analysis of two Pangolin species Manis pentadactyla and Manis javanica'. GigaScience Database.</p>
<p> </p>

[STEPS]
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TITLE: Protocols for "Sequencing smart: De novo sequencing and assembly approaches for a non-model mammal"
AUTHORS: Graham J Etherington, Darren Heavens, David Baker, Ashleigh Lister, Rose McNelly, Gonzalo Garcia, Bernardo Clavijo, Iain Macaulay, Wilfried Haerty, Federica Di Palma

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Whilst much sequencing effort has focused on key mammalian model organisms such as mouse and human, little is known about the correlation between genome sequencing techniques for non-model mammals and genome assembly quality. This is especially relevant to non-model mammals, where the samples to be sequenced are often degraded and low quality. A key aspect when planning a genome project is the choice of sequencing data to generate. This decision is driven by several factors, including the biological questions being asked, the quality of DNA available, and the availability of funds. Cutting-edge sequencing technologies now make it possible to achieve highly contiguous, chromosome-level genome assemblies, but relies on good quality high-molecular-weight DNA. Here we use a range of different genomic technologies generated from a roadkill European Polecat (Mustela putorius) to assess various assembly techniques on this low-quality sample. We evaluated different approaches for de novo assemblies and discuss their value in relation to biological analyses. The high degree of variability between each de novo assembly method (assessed from the seven key metrics) highlights the importance of carefully devising the sequencing strategy to be able to carry out the desired analysis. Adding more data to genome assemblies does not always results in better assemblies so it is important to understand the nuances of genomic data integration explained here, in order to obtain cost-effective value-for-money when sequencing genomes.</div></div>

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Conflicting results have been obtained regarding the association between X-ray repair cross complementation group 1 (XRCC1) and susceptibility to hepatocellular carcinoma (HCC). In this study, associations between HCC and three polymorphisms (Arg194Trp, Arg280His, and Arg399Gln) were evaluated using a meta-ananlysis approach. PubMed, Web of science, Cochrane Library, the Chinese National Knowledge Infrastructure, and the Wanfang standard database were systematically searched to identify all relevant case-control studies published through March 2018. A total of 32 case-control studies, including 13 that evaluated Arg194Trp, 14 that evaluated Arg280His, and 26 that evaluated Arg399Gln, were analyzed. In the entire study population, XRCC1 Arg399Gln was significantly associated not only with the overall risk of HCC (homozygous model, OR = 1.53, 95% CI: 1.34–1.76, P = 0.00; recessive model, OR = 1.31, 95% CI:1.15–1.49, P = 0.00), but also with the risk of HCC in Chinese patients (homozygous model, OR = 1.78, 95% CI: 1.53–2.08, P = 0.00; recessive model, OR = 1.47, 95% CI: 1.27–1.70, P = 0.00). Limiting the analysis to studies demonstrating Hardy–Weinberg equilibrium (HWE), the results were consistent and robust. Similarly, a significant association between XRCC1 Arg399Gln and HCC risk was found in healthy controls in the general population but not in hospital controls. Trial sequential analysis (TSA), the false-positive report probabilities (FPRP) and the combined genotype analysis revealed that XRCC1 Arg399Gln is mainly associated with susceptibility to liver cancer. However, there was no association between Arg194Trp or Arg280His and the risk of HCC. These results, indicating that the Arg399Gln polymorphism of XRCC1 is associated with the risk of HCC in the Chinese population, provide a basis for the development of improved detection and treatment approaches.</p>

[STEPS]
TITLE: Hi-C library construction from young Maize leaves
AUTHORS: Yikun Zhao, Yuancong Wang, De Ma, Guang Feng, Yongxue Huo, Zhihao Liu, Ling Zhou, Yunlong Zhang, Liwen Xu, Liang Wang, Han Zhao, Jiuran Zhao, Fengge Wang

[DESCRIPTION]
In a recent study we constructed a high-quality chromosome-level reference genome for the maize cultivar Dan340 by combining PacBio long HiFi sequencing reads, Illumina short reads and chromosomal conformational capture (Hi-C) sequencing reads. 

A Hi-C library was constructed using young leaves following previously published procedures with slight modifications. See the source paper and the following protocol.

 


[STEPS]
1. Approximately5 gleaf samples from seedling were cut into minute pieces and cross-linked by 4% formaldehyde solution at room temperature in a vacuum for30 min.

2. Each sample was mixed with an excess of 2.5 Molarity (M) glycine to quench the crosslinking reaction for 5 min and then placed on ice for15 min.

3. The cross-linked DNA was extracted and then digested for 720 min with 20 units of DpnII restriction enzyme (NEB) at 37 °C, and the resuspended mixture was incubated at65 °C for20 min to inactivate the restriction enzyme.

4. The sticky ends of the digested fragments were biotinylated and proximity ligated to form enriched ligation junctions and then ultrasonically sheared to a size of 300 - 600 bp.
5. The biotin-labelled DNA fragments were pulled down and ligated with Illumina paired-end adapters, and then amplified by PCR to produce the Hi-C sequencing library.
6. The library was sequenced using an Illumina HiSeq X Ten platform with 2 × 150 bp paired-end reads (Illumina, San Diego, CA, USA).
7. After removing low-quality sequences and trimming adapter sequences, we had 304.37 Gb (approximately 130×) of clean data generated. This is then used for genome assembly.
TITLE: &#39;Uniclear&#39; water-based brain clearing for light sheet imaging of electrode tracks
AUTHORS: Susu Chen, Karel Svoboda

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">UniClear procedure for whole mouse brain clearing and refractive index matching. The advantages of this method are that the method is water-based and produces a mechanically robust specimen. Cleared brains can be imaged with lightsheet microscopes (e.g. Zeiss Z1 lightsheet).  </div></div>

[STEPS]
?. Perfuse mouse with cold 4% PFA. Post fix brain for at least  at room temperature (RT). Wash brain in PBS to remove fixative. Fixed brain samples can be kept in PBS at  before further processing.
4 °C
?. Delipidate brain with SBiP buffer:  per adult mouse brain, shaking (70 RPM) at . On the first day: change buffer at , , and leave over night. Starting on the second day: change SBiP buffer every  for consecutive 14 days , shaking (70 RPM) at .
20 ml
37 °C
37 °C
?. After delipidation, wash brain in PBS for at least  before further processing. Sample can be stored in PBS at RT before refractive index matching.
?. Refractive index matching: first day in  50% PBS + 50% opti-prep mixed solution, rotating at RT for . On second day transfer the brain to  100% opti-prep solution, rotating at RT for . Index-matched brain should appear transparent.
20 ml
40 ml
?. For light-sheet imaging (e.g. Zeiss Z1) mount brain vertically. To image tissue autofluorescence use 488 nm laser. To image CM-Dil tracks use 561 nm laser. Brain can be imaged with a voxel size of 1.22 µm x 1.22 µm x 8 µm on Z1 with a 5x objective.