eamag/protocols-with-steps huggingface dataset:
TITLE: Quantitative PCR analysis to assess gene expression changes in hyperglycemic larval zebrafish
AUTHORS: Sandra Rieger

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary: </span></div><div class = "text-block"><span>To assess hyperglycemia in larval zebrafish, we analyzed the expression of genes implicated in glucose metabolism, such as insulin, insulin receptor, glucagon, and phosphoenolpyruvate carboxylase (pepck), using quantitative PCR (qPCR). We analyzed gene expression following treatment of Tg(</span><span style = "font-style:italic;">ins:NTRmCherry</span><span>) transgenic larvae either with 0.5 % DMSO (controls) or 10 mM metronidazole (see Protocol 2) for β-cell ablation at 3 and 8 days post fertilization (dpf). </span></div><div class = "text-block"><span style = "font-weight:bold;">Diabetic Complication:</span></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/pr-journal/bhzq4kcn.jpg" /></div></div>

[STEPS]
?. Preparation of mRNA from larval zebrafish. Pool 10 zebrafish larvae and isolate RNA according to the RNeasy Mini Kit (QIAGEN) manual. Elute final mRNA in 30 μl of RNase-free water.
?. Preparation of cDNA from mRNA. Prepare cDNA from isolated mRNA according to the SuperScript®III First-Strand Synthesis System (Life Technologies) using either oligo(dT) or random hexamer primers.
?. Preparation of qPCR mix (according to the Agilent manual for preparation of the Brilliant III Ultra- Fast SYBR® Green QPCR Master Mix):Dilute the reference dye 1:500 using nuclease-free PCR-grade water. Prepare the experimental reactions by combining the components of the reagent mixture in the order listed in the table below. Prepare a single reagent mixture for replicate reactions (plus at least one reaction volume excess) using multiples of each component.Reagent Mixture
Nuclease-free PCR-grade water to bring final volume to 20 μl (including cDNA) 10 μl of 2× SYBR Green QPCR Master Mix
x μl of upstream primer at optimized concentration (200–500 nM)
x μl of downstream primer at optimized concentration (200–500 nM)
0.3 μl of diluted ROX reference dye
Gently mix the reagent mixture without creating bubbles, then distribute the mixture to the experimental reaction tubes. Add 0.5-50 ng (x μl) of cDNA to each reaction to bring the final reaction volume to 20 μl.We amplified each transcript using the following PCR conditions:1) 95ºC 3 minutes2) 40 cycles    95ºC 15 seconds    60ºC 20 seconds,3) Melting curve    95ºC 1 minute    60ºC 30 seconds    95ºC 30 seconds
?. [Potential Pitfalls:]
Inconsistent qPCR results: Always use master mixes and the suggested reference dye (ROX reference dye, Life Technlogies, USA). Include pre-mix of triplicate reactions with sample and primers. Alternatively, utilize the Taqman system (Life Technologies).
TITLE: Trophoblast Organoid Media (TOM)
AUTHORS: vfarmer

[DESCRIPTION]
Media is also used with flipped TOs. Media can be stored at 4C and used within 1 month.

[STEPS]
SECTION: Recipe
1. In a 50 mL conical add 40 mL of F12/DMEM. Mix in the following components:
SECTION: Recipe
1.1. B27 1 mL

stock is 10X, final is 1X

supplier: ThermoFisher (Gibco), cat#: 17504044
SECTION: Recipe
1.2. N-Acetyl-L-Cystine (NAC) 625 µL

stock is 100 mM in H20, final is 1.25 mM

supplier: Sigma, cat#: A9165

SECTION: Recipe
1.3. N2 500 µL

stock is 10X, final is 1X

supplier: ThermoFisher (Gibco), cat#: 17502048
SECTION: Recipe
1.4. L-Glutamine 500 µL

stock is 200 mM, final is 200 mM

supplier: ThermoFisher (Gibco), cat#: A2916801
SECTION: Recipe
1.5. Nicotinamide (NTM) 500 µL

stock is 1 M in H20, final is 10 mM

supplier: Sigma-Aldrich, cat#: N0636
SECTION: Recipe
1.6. Primocin 100 µL

stock is 50 mg/ml, final is 100 µg/ml

supplier: Inviogen, cat #: NC9141851
SECTION: Recipe
1.7. Y-27632 100 µL

stock is 2.5 mM in H20, final is 5 µM
SECTION: Recipe
1.8. hEGF 25 µL

stock is 100 µg/ml in H20, final is 50 ng/ml

supplier: Peprotech, cat#: AF-100-15
SECTION: Recipe
1.9. hFGF2 25 µL

stock is 200 µg/ml in 5mM Tris pH 7.6, final is 100 µg/ml

supplier: Peprotech, cat#: 100-18C
SECTION: Recipe
1.10. hHGF 25 µL

stock is 100 µg/ml in H20, final is 50 ng/ml

supplier: Peprotech, cat#: 100-39
SECTION: Recipe
1.11. R-spondin1 25 µL

stock is 160 µg/ml in PBS+0.1% BSA, final is 80 µg/ml

supplier: Bio-techne, cat#: 4645-RS
SECTION: Recipe
1.12. PGE2 25 µL

stock is 5 mM in acetone, final is 2.5 µM

supplier: Sigma, cat#: P0409
SECTION: Recipe
1.13. A83-01 5 µL

stock is 5 mM in DMSO, final is 500 nM

supplier: Tocris, cat#: 2939
SECTION: Recipe
1.14. CHIR99021 5 µL

stock is 15 mM in DMSO, final is 1.5 µl

supplier: Tocris, cat#: 4423
SECTION: Recipe
1.15. FBS 5 mL

supplier: Cytiva, cat# SH30088.03HI
TITLE: The PacBio libraries preparation for the Scapharca broughtonii.
AUTHORS: Chang-Ming Bai

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol is used to outline the process of the PacBio libraries preparation for the </span><span style = "font-style:italic;">Scapharca broughtonii</span><span style = "font-style:italic;">.</span></div></div>

[STEPS]
?. The genomic DNA was fragmented using a g-TUBE (Covaris, Brighton, UK) to obtain ~20kb frequments, and verified with Agilent Bioanalyzer 2100 system  (Agilent Technologies, Palo Alto, CA, USA).
The sizes of the main fragments should >17 Kb.
?. End-repair and adaptor-ligation were carried out using SMRTbell Template Prep Kit (Pacific Biosciences, p/n 100-259-100). The precedures was keeped at 37℃, 60 min for damage repair; 25℃, 10 min for end repair, and 25℃, over night for adapter ligation.
?. These fragments were firstly selected into size ranges of 12-50 kb using BluePippin Size-Selection system  (Sage Science, Beverly, the U.S.), and further purified using AMPure PB beads (Agencourt, Beverly, the U.S.).
?. The purified fragments were re-repaired with SMRTbell Damage Repair Kit (Pacific Biosciences, p/n 100-465-900), and purified using AMPure PB beads.
?. The concerntration and size were detected with Qubit 3.0 (Thermo Fisher Scientific Inc., Carlsbad, CA, USA) and Agilent Bioanalyzer 2100 system  (Agilent Technologies, Palo Alto, CA, USA).
?. The qualified fragments were combined with primers, polymerase and Magbeads (Pacific Biosciences, p/n 100-676-500) using PacBio Bingding Kit (Pacific Biosciences, p/n 100-372-700), and ready for sequencing.
TITLE: 384 Well PicoGreen
AUTHORS: Allen Institute for Brain Science

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Quant-iT™ PicoGreen® dsDNA Assay is used for detection and quantitation of double stranded DNA products. </div><div class = "text-block"><span style = "font-weight:bold;">Note</span><span>: Research reported in this publication was supported by the National Institute Of Mental Health of the National Institutes of Health under Award Number U19MH114830. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.</span></div></div>

[STEPS]
TITLE: Collection of rat vagal tissue samples for TEM imaging
AUTHORS: Terry  Powley, Deborah  Jaffey

[DESCRIPTION]
This protocol describes the methods used to generate samples of rat vagus tissue suitable for TEM imaging.

[STEPS]
SECTION: Animals
1.   
rats (2–4 months old, male and female) were housed in shoe-box cages with bedding material in an Association for Assessment and Accreditation of Laboratory Animal Care-approved colony room, temperature (22–24°C) and humidity (40%–60%) controlled. The room was maintained on a 12:12 hour light–dark schedule. Pelleted chow
   
and filtered tap water were provided ad libitum. All husbandry practices conformed to the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals (8th edition) and were reviewed and approved by the Purdue University Animal Care and Use Committee. All efforts were made to minimize any suffering as well as the number of animals used.

SECTION: Dissection and Fixation
3. Nerve bundles, trunks, and branches were immediately dissected out, manipulating the vagus as little as possible, removing only the minimum amount of connective tissue or fat require to access and remove the samples (4mm long sections at each sampled level) and moved into the same fixative combination as in step 2 overnight at 4°C on an oscillating platform.

The following morning, the tissue specimens were rinsed (3 × 30 min at 4°C) in PBS, transferred to individual shipping vials under PBS, and shipped overnight to the TEM laboratory for further processing and microscopy - see the protocol below:
 

SECTION: Perfusion
2. Animals were perfused according to the following procedure. Specifically, they were overdosed with anesthetic (intraperitoneal injection of ketamine/xylazine [275 mg/kg of ketamine/27.5 mg/kg of xylazine]):
 
  
Animals were then exsanguinated with fresh physiological saline, and then transcardially perfused with a fresh solution of EM-grade fixatives (2% paraformaldehyde/1.25% glutaraldehyde) at 4°C:
  
  
 in 0.1 M phosphate-buffered saline ([PBS], pH = 7.3) for 20 minutes.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
This is the "quick" version of Monarch® PCR &amp; DNA Cleanup Kit (5 μg) Protocol (NEB #T1030). For the full protocol, please click <a href="https://www.neb.com/protocols/2015/11/23/monarch-pcr-and-dna-cleanup-kit-protocol" target="_blank">here</a>.

[BEFORE_START]
Add 4 volumes of ethanol (≥ 95%) to one volume of DNA Wash Buffer.<br />
<ul>
<li>For 50-prep kit, add 20 ml of ethanol to 5 ml of Monarch DNA Wash Buffer</li>
<li>For 250-prep, kit add 100 ml of ethanol to 25 ml of Monarch DNA Wash Buffer</li>
</ul>
All centrifugation steps should be carried out at 16,000 x g (~13,000 RPM).

[GUIDELINES]
For detailed protocol and more information, visit <strong><a href="https://www.neb.com/products/t1030-monarch-pcr-dna-cleanup-kit-5-ug" target="_blank">www.neb.com/T1030</a></strong><br /><br />The full protocol is available <a href="https://www.neb.com/protocols/2015/11/23/monarch-pcr-and-dna-cleanup-kit-protocol" target="_blank">here.<br /><br /></a>The video protocol is available <a href="https://www.neb.com/tools-and-resources/video-library?videoid=%7B0d9e96c2-2bfa-47a7-babc-b5e584213db7%7D" target="_blank">here</a>.<br /><a href="https://www.neb.com/products/t1030-monarch-pcr-dna-cleanup-kit-5-ug" target="_blank"></a>

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
This protocol gives a method for prophage induction in marine <em>Synechococcus.<br /><br /></em>Paul, J. H., and M. Weinbauer. 2010. Detection of lysogeny in marine environments, p. 30–33. In S. W. Wilhelm, M. G. Weinbauer, and C. A. Suttle [eds.], Manual of Aquatic Viral Ecology. ASLO.<em><br /></em>

[BEFORE_START]
The samples for prophage induction are pretreated by the technique of viral reduction (Weinbauer and Suttle 1996).

[GUIDELINES]
<strong>Materials</strong> <br /><br />Sterile 96 well microtiter plates <br />Indicator host culture (i.e., Synechococcus WH7803)<br /><br /><strong>References<br /><br /></strong>Hurley, M. A., and M. E. Roscoe. 1983. Automated statistical analysis of microbial enumeration by dilution series. J. Appl. Bacteriol. 55:159-164.<br /><br />Suttle, C. A., and A. M. Chan. 1994. Dynamics and distribution of cyanophages and their effect on marine Synechoccus spp. Appl. Environ. Microbiol. 60:3167-3174.<strong><br /><br /></strong>Weinbauer, M. G., and C. A. Suttle. 1996. Potential significance of lysogeny to bacteriophage production and bacterial mortality in coastal waters of the Gulf of Mexico. Appl. Environ. Microbiol. 62:4374-4380.<strong><br /></strong>

[STEPS]
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TITLE: No Title
AUTHORS: 

[GUIDELINES]
<strong>Materials</strong><br /><br />1) L broth<br />
<ul>
<li>1.0% Bactro-tryptone</li>
<li>0.5% Bacto-yeast extract</li>
<li>0.5% NaCl</li>
</ul>
After autoclaving, add 1.0 mL of 20% glucose per 100 mL of L broth<br /><br />2) 10mM Tris-HCl, pH 8.0, 50 mM CaCl<sub>2<br /><br /></sub>3) L broth + antibiotic (of the plasmid transformed)<br /><br /><strong>Reference</strong><br /><br />Maniatis, E.F. Ftitsch, and J. Sambrook. (1982).  <u>Molecular</u> <u>Cloning:  A Laboratory </u><u>Manual.</u>  Cold Spring Harbor Laboratory.  Pages 250-251.<u><br /></u>

[STEPS]
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TITLE: DRBUDDI for MRI Processing
AUTHORS: Courtney Comrie

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol will provide a basic guide to utilizing the DRBUDDI tool in TORTIOSE.</div><div class = "text-block">Note: Steps may vary based upon image.</div></div>

[STEPS]
?. [Introduction]
DRBUDDI is a EPI distortion correction module in TORTOISE. It bases its corrections by using DTI data pairs (blip up and blip down). When processing MR images DRBUDDI is usually the second step in the pipeline.
?. [DRBUDDI]
In terminal go to your directory with the DTI data.
?. [DRBUDDI]
Type the following in the terminal to view information
?. [DRBUDDI]
In terminal type:
?. [DRBUDDI]
You should have two corresponding data sets to edit, and you will need to distinguish them when you call DRBUDDI i.e. blip up and blip down
?. [DRBUDDI]
NOTE: There are two DRBUDDI versions available to use. There is a DRBUDDI with a GUI, and there is one without a GUI.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>The GF-1 Soil Sample DNA Extraction Kit is designed for the rapid and efficient purification of bacteria DNA from soil samples without the need for precipitation or organic extraction. The kit uses a high pure specially-treated silica-based material fixed into a column to efficiently bind DNA in the presence of high salt. The kit applies the principle of a minicolumn spin technology and the use of optimized buffers to ensure that only DNA is isolated while cellular protein, humic acid, metabolites, salts and other low molecular weight impurities are removed during the subsequent washing steps. We used this protocol for <em>Cryptosporidium</em> spp DNA extraction from stool samples with minor modifications and its working.</p>

[BEFORE_START]
<p>Pre-set the water bath at 70<sup>o</sup>C.</p>
<p> </p>

[GUIDELINES]
<p>All steps should be carried out at room temperature unless stated otherwise.</p>

[STEPS]
?. 
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TITLE: Stranded Mapping from Oriented Long Reads
AUTHORS: David A Eccles

[DESCRIPTION]
This protocol demonstrates how to map strand-oriented long reads to a genome, and visualise them in a genome browser.
 
The general idea is to use minimap2 to create stranded BAM files, which are split for forward/reverse orientation then converted into BigWig format for display in a genome browser.

Input(s): 
stranded fastq files (see protocol Preparing Reads for Stranded Mapping)
a FASTA file containing the genome / sequence of interest.
 
Output(s):
Genome-mapped stranded BAM files
Genome-mapped stranded BigWig files

[BEFORE_START]
You will need access to the following free and open-source software program(s):
minimap2
samtools

And the following additional data file(s):
a FASTA file containing the genome / sequence of interest.

[STEPS]
SECTION: Orient Reads
1. Orient reads as per protocol Preparing Reads for Stranded Mapping.

If this has been done, then the following command should produce output without errors:
 
Example output:
 


SECTION: Index Preparation
2. Prepare genome index for spliced alignment:
 

 

Prepare a chromosome size index file:

 


SECTION: Read Mapping
3. Map the long reads to the genome using minimap2, using samtools to covert to a sorted BAM format. This is where the reverse complementing done during demultiplexing gives a big saving of effort. As this BAM file is one of the main outputs, the run name is added to the file name.

 

 


SECTION: Creating BigWig Coverage Files (not needed for JBrowse 2)
4.  
A bedGraph of coverage is created using BEDTools. The default options for BEDTools treat sequence deletions (which happen frequently in nanopore reads) as a drop in coverage, which can make exon hunting and coverage calculation more difficult. I submitted a pull request to add an additional ignoreD parameter to the command line to allow cDNA reads with split coverage across introns to ignore deletions when considering coverage; this request has now been incorporated into the main BEDtools repository (as of v2.30.0):
 


 

SECTION: Creating BigWig Coverage Files (not needed for JBrowse 2)
5. Stranded bedgraph files are converted to bigwig. This requires BEDTools and a genome information file containing chromosome lengths, as generated in the first step:

 


SECTION: JBrowse 2 Configuration
6. 
 

Each BAM file should have its own JBrowse rack. JBrowse 2 can create both alignment views and coverage plots from BAM files:

 

If you have a "smart" file system mount that doesn't let you directly copy files via the jbrowse add-track command, you can use the /tmp file system and symlinks to do the same thing:


 

SECTION: Sanity Check
7. If this has worked properly, then mapping human or mouse to the mitochondrial genome with a LinearSNPCoverageDisplay in log scale should show a stepped expression, a bit like the Expected Results shown here:
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p><strong>Note: If the percentage of CD45+ cells in your sample is less than 50%, please follow Protocol A. If it is higher than 50% then please follow protocol B.</strong></p>
<p> </p>
<p>The cells targeted by the Nanobeads are either selected or depleted by incubating your sample with the directly conjugated magnetic particles. The magnetically labeled fraction is retained by the use of a magnetic separator. After collection of the targeted cells, downstream applications include functional assays, gene expression, phenotypic characterization, etc.</p>

[STEPS]
?.
TITLE: Plasma Exosome Isolation
AUTHORS: Silvia Cerri

[DESCRIPTION]
This protocol details methods for the isolation of small extracellular vesicles from plasma. 

[BEFORE_START]
Keep samples on ice during the entire procedure!


[STEPS]
SECTION: Plasma Exosome Isolation
1. Incubate plasma samples (starting volume: 1-1.5ml) on ice for complete thaw.

SECTION: Plasma Exosome Isolation
2. Filter samples through a 13mm sterile syringe filters with a 0.8 µm Supor (PES) membrane.
SECTION: Plasma Exosome Isolation
3. Centrifuge at 20000 x g, 60 min, 4 °C to remove large extracellular vesicles.

SECTION: Plasma Exosome Isolation
4. Collect the supernatant and filter it through 13mm sterile syringe filters with 0.2 mm Supor (PES) membrane.
SECTION: Plasma Exosome Isolation
5. Centrifuge at 100000 x g, 60 min, 4 °C to pellet small extracellular vesicles.

SECTION: Plasma Exosome Isolation
6. Remove the supernatant, resuspend the pellet in 1 mL and repeat the previous step (repeated again in the next step for convenience).
 

SECTION: Plasma Exosome Isolation
8. Process the pellet according to the type of analysis.
SECTION: Plasma Exosome Isolation
7. Centrifuge at 100000 x g, 60 min, 4 °C to pellet small extracellular vesicles.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This method describes the comparison of paracetamol quantification of a whole tablet vs a tablet that has been crushed in a proprietary tablet crusher. In this case we have used paracetamol as the model drug, but this basic approach can be applied to any solid dose form with appropriate adjustment of solvents (to ensure drug is completely dissolved for measurement) and UV wavelength. The use of HPLC can be substituted in place of simple UV if there is any indication of interference at the UV wavelength.</p>

[STEPS]
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TITLE: RNA extraction from diatom P. multistriata 
AUTHORS: Francesco   Manfellotto, Antonella  Ruggiero, Pina  Marotta, Monia Teresa  Russo, Anna  Santin, Mariella  Ferrante

[DESCRIPTION]
RNA extraction protocol from diatom P. multistriata

[STEPS]
1. Collect 20/40 million cells
2. Harvest cells by filtration onto 1.2 µm pore size filter

3. Cut the filter into two halves and store each half in separate 2 mL eppendorf tube.
4.  Add 1 mL of TRIzol® Reagent (for 5-10 × 10^6 cells) to eppendorf with half filter and vortex briefly. 
  

5. Flash freeze the eppendorf immediately in liquid nitrogen. 
The eppendorf can be stored at -80 °C for later extraction. 

Note: Do not wash cells before addition of TRIzol® Reagent.

Defrost the samples at room temperature (use thermoshaker for 15 sec at 60 °C if defrosting takes longer). 


DO NOT PROCESS MORE THAN 6 SAMPLES AT SAME TIME.
6. Add 0,2g glass beads and put on thermoshaker at 60 °C for 10 min with maximum rpm (1400).  

7.  Centrifuge briefly for one minute to get rid of beads and filter and move the supernatant into 2 mL eppendorf tube.
8. Add 200 ul of CHLOROFORM per 1 ml of Trizol
11. Centrifuge the eppendorf for 15 minutes at 10600 rpm (12,000 g) AT MAXIMUM , at 4 °C.
12. Transfer 500 ul (half the volume of Trizol used originally) of uppermost aqueous layer (contains RNA) into new 2mL eppendorf. 

Do not disturb middle colorless phase and lowermost pink phase while pipetting (these phases contain proteins and DNA).
13. Add equal volume of ISOPROPANOL (500 ul) and invert the tube to mix. 

Don’t shake. 

14. Incubate at room temperature for 10 minutes 

(samples can be stored at 4 °C overnight).
15. Centrifuge for 10 minutes at 10600 rpm at 4 °C. 
16. Remove supernatant leaving RNA pellet undisturbed.
17.  Add 1 mL of 75% ethanol. Invert the tube gently to wash the pellet 

(samples can be stored at -20 °C for long time).
18.  Centrifuge for 10 minutes at 8500 rpm (7500 g) at 4 °C. 
19. Remove supernatant leaving RNA pellet undisturbed.
20. Allow the pellet to dry for 20-30 minutes to remove all the traces of ethanol. 


Care should be taken not to over dry the pellet.
21.  Re-suspend the pellet in 20-30 ul of RNA-se free water and dissolve the pellet by pipetting up and down.


For better re-suspending, incubate the samples for 5-10 minutes at 55-60 °C.
9. Shake the eppendorf vigorously for 15 seconds
10. Incubate for 15 minutes at room temperature.
TITLE: Green Lab Nanoparticle For 6 Well Cardiomyocyte Transfection v2
AUTHORS: Edwin Yoo

[DESCRIPTION]
nanoparticle transfection    

[STEPS]
SECTION: Nanoparticle Synthesis
2. DNA Dilution
For 3 technical replicate wells of one condition in 6-well plate format, mix the following in microcentrifuge tube.
60 ul DNA at 1ug/ul
440 ul sodium acetate
Total volume = 500 ul for 3 technical replicate wells per one condition
SECTION: Nanoparticle Synthesis
3. Polymer Dilution (7.2 mg.ml polymer concentration in NaAc)
For 3 technical replicate wells of one condition in 6-well plate format, mix the following in microcentrifuge tube.
40 ul polymer (e.g. 4-5-6)    
460 ul sodium acetate
Total volume = 500 ul for 3 technical replicate wells per one condition 
SECTION: Nanoparticle Synthesis
4. Nanoparticle (DNA+polymer) Synthesis
Combine diluted DNA with diluted polymer
Add 500 ul of diluted DNA to 500 ul diluted polymer (can freeze here at -80 at your own risk)
Add 5000 ul of serum containing media to 1000 ul NP's for final volume master mix of 6000 ul per condition
Incubate for 10 minutes
SECTION: Nanoparticle Synthesis
5. Transfection
Change media from each well of 6 well plate with 2000 ul nanoparticle master mix (media + nanoparticles)
Incubate for 24 hours or desired time
Change media after desired transfection time.
SECTION: Nanoparticle Synthesis
1. Defining Terms
DNA refers to nucleic acid cargo encoding genes of interest, generally stored at a concentration of 1 ug/ul.
Polymer refers to the Poly(beta-amino ester) (PBAE) that makes up the backbone of the nanoparticle.
Nanoparticle refers to the final complexed polymer + DNA that is ready for transfection.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This single-tube library construction protocol is for degraded DNA using adapters for the Illumina platform.</p>

[BEFORE_START]
<p>Calculate the DNA input and prepare the library adapters.</p>

[STEPS]
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