Sample Preparation and Shipping

Eukaryotic Genome Sample Prep

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Eukaryotic Genome Sequencing with Extraction

Service Level	Size	Number of Cells	Preservative	Cost
1 Gb data target	20 - 60 Mb	1 x 10^6	200 µL Zymo DNA/RNA Shield™	$300
5 Gb data target	75 - 250 Mb	1 x 10^6		$550
15 Gb data target	300 - 750 Mb	2 x 10^6		$1050
Full flow cell (50 - 100 Gb data target)	1 - 3.3 Gb	2 x 10^6		$1800

Count your cells.

This service requires 1-2 x 10^6 cells (depending on the service level) that are preserved in Zymo 1X DNA/RNA Shield™. This service is available for most cultured or disassociated eukaryotic cell types that do not have a cell wall (including isolated protoplasts with their cell walls removed). We accept cell pellets from both BSL1 and BSL2 strains. If you have fewer cells available than listed above, we can often work with lower inputs too - please email us at support@plasmidsaurus.com to discuss options.

We do not currently accept blood from any species, nor any intact tissues or organs from animals, plants, insects, fungi, etc. We also do not accept cells preserved by other methods such as flash freezing, ethanol, desiccation, RNAlater, etc. If you’d like to sequence these alternative sample types and preservation methods, please perform extraction on your end and ship us only the purified gDNA for sequencing.

Wash and pellet cells.

Pellet the cells by centrifugation and remove as much supernatant as possible.

Resuspend the pellet in 1 mL PBS to wash, then pellet again and remove supernatant.

Resuspend the pellet.

Resuspend the final pellet in 200 µL of Zymo 1X DNA/RNA Shield™ in a 200 μL strip tube (when ordering Zymo DNA/RNA Shield™, you can enter PLASMIDSAURUS at checkout for 10% off).

Please do not use any other type of preservation media, as they do not preserve the DNA as effectively and do not fully inactivate the host cells, which is required for safe handling in our lab.

Label your tubes or plates.

Set up your order online and receive your unique 6-character order ID.
Write the first 2 characters of your order ID on the 1st tube of each strip

Set up your order online and receive your unique 6-character order ID. On the order page, please check the “Is this a plate submission?” checkbox, then select if your plate is numbered sequentially by column or by row.
Label the front edge of the plate with the full 6-character order ID.
If you are using column numbering, label the first column (i.e., samples 1 to 8). If you are using row numbering, label the first row (i.e., samples 1 to 12).

Load and package your sample.

Add the correct volume of your sample(s) from the table above. Make sure to seal and package your samples well to make sure they arrive safely for sequencing!

Ensure caps fit snugly and close them firmly. Do not use brands of strip tubes where the caps can easily pop open during shipping.
Place the labeled tubes into a small protective vessel (such as a Falcon tube, a small box, or bubble wrap) to protect them during shipping. Why?

Start with well A1 and load your samples contiguously. Our preference is for samples to be loaded by column but we can accept samples loaded by row if indicated in the order. Do not leave any empty wells between samples.
Ensure your plates are sealed extremely thoroughly to survive shipping without leaking — sadly we receive a lot of leaky plates. We recommend using a heat seal (rather than adhesive) or snugly-fitting strip caps on top of the plate. If you only have adhesive options, click here for some examples of sealing technique.
To prevent cross contamination of samples, our customers have also had good success with dehydrating plates: spin the plate(s) down and then place the plate(s) on a 65 degree heating block for 0.5 to 1 hour or until dry. After dry, you can seal the plate with sticky film to prevent contamination of the sample during transport. We will rehydrate the sample in our lab for sequencing.
Wrap each plate individually in bubble wrap or other packaging material to prevent cracking and seal punctures. DO NOT stack multiple plates directly on top of each other, as the wells of one plate will likely puncture the seal of the next plate.

Dropoff or ship.

Print out your order form. Fold so that QR code is readily visible and place in a small bag with your samples.

For fastest results and easiest shipping, place your samples in a Plasmidsaurus Dropbox!

If you ship your samples to us directly:

Place packaged samples in a sturdy cardboard or padded envelope or a cardboard box. If reusing old packaging, ensure that any hazard warnings are completely obliterated. Multiple orders can be shipped in the same package, as long as each order is placed into a separate bag with its corresponding order confirmation sheet.
Do not add ice packs or dry ice. Samples should be shipped at room temperature.
Ship via any Express or First-Class shipping carrier such as FedEx, UPS, DHL, or USPS. The shipping address will be provided on your order confirmation sheet.

Please reach out to support@plasmidsaurus.com for specific guidance on international shipping, since the types of eukaryotic samples can vary greatly.

Eukaryotic Genome Sequencing without Extraction

Service Level	Size	Sequencing Platform	Normalized Concentration	Minimum Volume	Cost
1 Gb data target	20 - 60 Mb	ONT	50 ng/μL	20 μL	$250
5 Gb data target	75 - 250 Mb			40 μL	$500
15 Gb data target	300 - 750 Mb			60 μL	$1000
Full flow cell (50 - 100 Gb data target)	1 - 3.3 Gb			60 μL	$1750

Extract and purify genomic DNA.

Use your favorite method to extract and purify genomic DNA from your eukaryotic tissue or cells. RNase treatment is required during extraction.

Any method that yields high quality, high purity, high molecular weight (HMW), double-stranded gDNA that is free of nicks, gaps, breaks, and contaminants is suitable. Our favorite is the NEB Monarch® Spin gDNA Extraction Kit.

Be gentle with your gDNA

Avoid vortexing and fast or unnecessary pipetting; pipet with wide-bore tips only
Elute in elution buffer, not water
Do not expose to high temperatures (>37°C) for >1 hour, pH extremes ( <6 or>9), intercalating fluorescent dyes, or UV radiation
Avoid freeze-thaw cycles; store gDNA at 4°C for 1-2 months
If using a speed-vac, avoid over-drying of gDNA; do not use heat in the speed-vac

Samples should NOT contain any of the following

Sequencing primers
RNA (RNase treatment is required during extraction)
Denaturants (guanidinium salts, phenol, etc.) or detergents (SDS, Triton-X100, etc.)
Residual contaminants from the organism (heme, humic acid, polyphenols, etc.)
Insoluble material, colors, or cloudiness

Perform quality control checks.

Our low prices and fast turnaround times do not include gDNA quality control (QC) services, so it’s important that you verify that your samples meet our requirements prior to shipping them to us.

Measure gDNA concentration with fluorometry

We require performing DNA quantification assay with Qubit or equivalent fluorometric method such as a plate reader. Spectrophotometric methods like Nanodrop are much less accurate and submitting the wrong concentration can cause sequencing failure!
- Please note: High molecular weight (HMW) gDNA often requires extra homogenization effort (longer incubation time, increased incubation temperature, very extensive gentle mixing, etc.) to obtain accurate quantification. If separate DNA quantifications from the top and bottom of the sample are within 15% of each other, this is usually a good indication of adequate homogeneity.

Measure gDNA purity with spectrophotometry

This step is strongly recommended. We recommend gDNA samples with 260/280 of ~1.8 and 260/230 between 2.0-2.2. Purity assay may be performed with Nanodrop or other spectrophotometric methods. If your sample is contaminated and does not meet this metric, either re-extract the sample or clean up the sample to remove the contaminants using a Qiagen cleanup kit or AMPure XP beads.

Assess gDNA quality with gel electrophoresis

This step is strongly recommended. Get the most out of long-read sequencing by keeping your fragments long. We recommend gDNA samples with >50% of the total DNA mass contained within fragments above 15 kb in size. If the sample does not meet this metric, we strongly recommend repeating extraction. Size characterization may be performed with Femto Pulse, Fragment Analyzer, Bioanalyzer, or a slab gel with a HMW ladder (ideally with pulsed-field electrophoresis).

Normalize genomic DNA concentration.

Submit your final purified genomic DNA normalized to 50 ng/μL in the minimum volume of elution buffer (10 mM Tris, pH 8.5) that is specified in the table above.

If you don’t have enough DNA to reach the minimum volume at 50 ng/μL, we’d recommend extracting more DNA if possible. If that’s not possible, send us an email at support@plasmidsaurus.com and submit a lower volume at 50 ng/μL.