Tag Archives: mantle

Samples Received – C.gigas Tissue & DNA from Katie Lotterhos

Received 6 samples from Katie today. The box was labeled and stored @ -20C.

 

Here description of the samples, via email:

Lotterhos samples (gigas) arriving tomorrow

1) Mantle tissue samples of C. gigas were collected on 20140705 (source: Pipestem Inlet) by KEL
2) Extraction on 20141028 by VG using Qiagen DNAeasy Blood and Tissue Kit
3) Beadwash on 20150720 by VG using homemade sera-mag speed beads
4) Qubit 3.0 quantification on 20151206 by KEL and the following amounts were sent:

M1: 13 uL of 386 ug/mL
M2: 13.8 uL of 326 ug/mL
M3: 13.15 uL of 380 ug/mL (solution looked cloudy)

 

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DNA Isolation – Olympia Oyster Outer Mantle gDNA

Isolated additional gDNA for the genome sequencing. To try to improve the quality (260/280 & 260/230 ratios) of the gDNA, I added a chloroform step after the initial tissue homogenization.

Used 123mg of Ostrea lurida outer mantle collected by Brent & Steven on 20150812.

  • Homogenized in 500μL of DNAzol.
  • Added additional 500μL of DNAzol.
  • Centrifuged 12,000g, 10mins, @ RT.
  • Split supernatant equally into two tubes.
  • Added 500μL of chloroform and mixed moderately fast by hand.
  • Centrifuged 12,000g, 10mins, RT.
  • Combined aqueous phases from both tubes in a clean tube.
  • Added 500μL of 100% EtOH and mixed by inversion.
  • Spooled precipitated gDNA and transferred to clean tube.
  • Performed 3 washes w/70% EtOH.
  • Dried pellet 3mins.
  • Resuspended in 200μL of Buffer EB (Qiagen).
  • Centrifuged 10,000g, 5mins, RT to pellet insoluble material.
  • Transferred supe to clean tube.

DNA was quantified using two methods: NanoDrop1000 & Qubit 3.0 (ThermoFisher).

For the Qubit, the samples were quantified using the Qubit dsDNA BR reagents (Invitrogen) according to the manufacturer’s protocol and used 1μL of sample for measurement.

Results:

Qubit Data (Google Sheet): 20151125_qubit_gDNA_geoduck_oly_quants

METHOD CONCENTRATION (ng/μL) TOTAL (μg)
Qubit 137 27.4
NanoDrop1000 295 59.0

 

Yield is solid. We should finally have sufficient quantities of gDNA to allow for BGI to proceed with the rest of the genome sequencing! Will run sample on gel to evaluate integrity and then send off to BGI.

The NanoDrop & Qubit numbers still aren’t close (as expected).

The addition of the chloroform step definitely helped improve the 260/280 OD ratio (see below). However, the addition of that step had no noticeable impact on the 260/230 OD ratios, which is a bit disappointing.

 

NanoDrop Absorbance Values & Plots

 

 

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Genomic DNA Isolation – Olympia oyster adductor musle & mantle

Previously isolated gDNA from these tissues on 20150901. However, found out after the isolations that BGI needs >73μg of gDNA for the genome sequencing project, which is significantly more than I obtained previously.

Isolated gDNA from Ostrea lurida (Olympia oyster) adductor muscle & mantle samples collected by Brent & Steven on 20150812 using DNAzol (Molecular Research Center) according to the manufacturer’s protocol, with the following adjustments:

  • 89.8mg of adductor muscle
  • 92.2mg of mantle
  • Tissues homogenized in 750μL of DNAzol with disposable mortar/pestle tubes using 10 pestle strokes
  • After homogenization, topped off tubes to 1000μL with DNAzol and incubated @ RT for 10mins.
  • Performed optional centrifugation step (10,000g, 10mins @ RT)
  • Initial pellet wash was performed using a 70%/30% DNAzol/EtOH
  • Pellets were resuspended in 200μL of Buffer EB (Qiagen)
  • Insoluble material was pelleted (12,000g, 10mins @ RT) and supe transferred to new tubes

Spec’d on Roberts Lab NanoDrop1000 (ThermoFisher) and stored temporarily at 4C to avoid freeze-thawing before sending off for sequencing.

 

Results:

 

There was a great deal of insoluble material from the get-go that was carried through the entire isolation.

Overall, the 260/280 ratios look pretty good, but the 260/230 ratios are just trash. As can be seen in the plots above, there is clearly significant absorbance in the 230 – 250nm, suggesting some contaminant carryover (phenol/salt).

Will evaluate gDNA integrity on agarose gel.

Total yield from this isolation is still below the minimum quantity of gDNA needed for the sequencing project. Will need to perform another round of gDNA isolation.

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DNA Quantification – Claire’s Sheared C.gigas Mantle Heat Shock Samples

I previously checked Claire’s sheared DNA on the Bioanalyzer to verify the fragment size and to quantify the samples. Looking at her notebook, her numbers differ greatly from the Bioanalyzer, possibly due to the fact that the DNA1000 assay chip used only measures DNA fragments up to 1000bp in size. If her shearing was incomplete, then there would be DNA fragments larger than 1000bp that wouldn’t have been measured by the Bioanalyzer. So, I decided to quantify the samples on the NanoDrop1000 (ThermoFisher) again.

 

Results:

Spreadsheet: 20150226_Claire_sheared_Emma_1000ppm_OD260s

 

 

 

Comparison of NanoDrop1000 and Bioanalyzer measurements.

Sample NanoDrop (ng/μL) Bioanalyzer (ng/μL)
2M sheared 48.03 16.28
4M sheared 190.96 58.52
6M sheared 141.56 42.98
2MHS sheared 221.93 32.45
4MHS sheared 257.48 43.82
6MHS sheared 202.02 51.12

The NanoDrop is known to overestimate sample quantities due to the indiscriminate nature of UV spectrophotometry and that could be the reason for the large discrepancy between the two measurements (i.e. RNA carryover may lead to overestimation). As such, I’ll quantify the samples using a fluorescence-based assay for double stranded DNA tomorrow in hopes of getting the most accurate measurement.

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Bioanalyzer – C.gigas Sheared DNA from 20140108

To complement MBD ChiP-seq data and RNA-seq data that we have from this experiment, we want to generate, at a minimum, some BS-seq data from the same C.gigas individuals used for the other aspects of this experiment.  Claire had previously isolated DNA and sheared the DNA on 20140108. If possible, we’d like to perform MBD enrichment, but the current quantities of DNA may prevent us from this.

To quantify the DNA and evaluate the shearing profile, I ran 1μL of each of the following mantle pre-/post-heat shock samples on a DNA 1000 chip (Agilent) on the Agilent 2100 Bioanalyzer. in the Seeb Lab:

M = mantle
HS = heat shocked

  • 2M sheared
  • 4M sheared
  • 6M sheared
  • 2M HS sheared
  • 4M HS sheared
  • 6M HS sheared

Results:

Bioanalyzer Data File (XAD): 2100_expert_DNA_1000_DE72902486_2015-02-19_11-32-35(2).xad

 

Electropherograms

2100 Bioanalyzer electropherograms of Claire’s sheared C.gigas DNA.

 

Spreadsheet: 2100 expert_DNA 1000_DE72902486_2015-02-19_11-32-35_Results_001

 

Claire’s notebook entry doesn’t ever specify what her target shear size was, but the Bioanalyzer analysis suggests an average size of ~500bp.

Also interesting to note is that Claire’s sample concentrations (as measured on the NanoDrop1000) are significantly greater than what is calculated by the Bioanalyzer. Since the Bioanalyzer chip used (DNA1000) only goes to 1000bp, is it possible the differences in concentrations is due to incomplete shearing of the samples (e.g. a significant portion of the DNA is >1000bp in size and thus not factored in to the Bioanlyzer concentrations calculations)?

Will check sample volumes and determine total amount of remaining DNA for each sample and then assess how to proceed next (i.e. MBD or just BS-seq).

UPDATE 20150226:

Sample volumes were measured and total quantity (ng) of DNA in each sample were added to the spreadsheet above.

Based on the quantities of DNA we have for each sample, will discuss sequencing options (e.g. MBD or not, self-prepare libraries or not, etc) with Steven.

 

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qPCR – C.gigas COX1/COX2 Tissue Distribution

Performed qPCR using pooled cDNA from 20110311. Pooled 2uL from each of the following samples groups: Dg 3hr C, Gill 1hr C, Gill 1hr E, Mantle 3hr C, and Muscle 3hr C. Master mix calcs are here. Plate layout, cycling params, etc can be found in the qPCR Report (see Results). Primers sets run were:

EF1_qPCR_5′,3′ (SR IDs: 309, 310)

Cg_COX1/2_qPCR_F (SR ID: 1192) + Cg_COX1_qPCR_R (SR ID: 1191)- Target = COX1

Cg_COX1/2_qPCR_F (SR ID: 1192) + Cg_COX2_454align1_R (SR ID: 1190) – Target = COX2

Results:

qPCR Report (PDF)

qPCR Data File (CFX96)

Graphs were generated using the BioRad CFX Manager v2.0 software. Expression was normalized to EF1. Also to note, gene efficiency was assumed as 100% by the software since no standard curve was run on the plate. As such, analysis of this data may not be exact.

It’s clear by examining the graphs that the primers being used to differentiate COX1 and COX2 (since they share a common primer: SRID 1192) are differentially expressed. This indicates that the primer sets are indeed amplifying different targets as hoped. This was the primary intention of this qPCR. However, we also now have an idea of tissue distribution of the two genes, as well as their response to V. vulnificus exposre after 1hr. Next step is to perform this qPCR on all the individuals from this experiment as well as the different tissues.

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DNase – DNase C.gigas RNA from 20110120, 20110121 and 20110124

5ug of RNA was DNased using Ambion’s Turbo DNA-free kit, following the rigorous protocol (0.5uL of DNase for 30 mins then additional 0.5uL of DNase for 30mins). Calcs for DNase reactions are here. RNA was stored @ -80C in Shellfish RNA Boxes 4 and 5. Samples will be spec’d on Monday.

Results:

Overall, the RNA looks really good (based on OD 260/280 numbers). Not surprisingly, the OD 260/230 values for all samples dropped, likely due to the addition of the buffer (salts) used in the DNase reaction. Emma says she will check these samples for residual DNA.

–UPDATE (20110131)– Emma checked all DNased RNA samples on 20110131 using C.gigas 18s primers (SR ID: ?). She has not listed the results of the whether or not all samples are clean or if some still have residual gDNA carryover.

–UDPATE (20110201– Samples that appear to have residual gDNA carryover based on Emma’s qPCR on 20110131: Muscle C6, Gill 1hr C2 & E2.

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RNA Isolation – Various C.gigas Tissue from 20110111

RNA was isolated in 1mL TriReagent, according to protocol. Samples were resuspended in 50uL 0.1% DEPC-H2O and spec’d. RNA was stored @ -80C in “Shellfish RNA Box #4

Results:

RNA looks OK. Not surprising, but mantle and Dg/Gonad tissues ended up with poor OD260/230 ratios. This has been observed in the past with these tissue types.

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qPCR – COX qPCR Primer Test and Tissue Distribution

Used new cyclooxygenase primers (SR IDs 1060, 1061) to see how they performed and to evaluate tissue distribution. Tissue distribution was evaluated using the following cDNAs made on 10/27/10 from Emma:

Gigas Digestive Gland

Gigas Gill

Gigas Mantle

Gigas Muscle

qPCR Master Mix calcs are here. Plate layout, cycling parameters, etc can be found in the qPCR Report (see Results).

Results:

qPCR Report (PDF).

Amplification is present in all four tissue types and the melting curve looks good. So, these primers are good to go. Steven suggests checking to see if we see a change in gene expression from an old experiment of Gigas exposed to high levels of Vibrio tubiashii. Will round up some old cDNA for this.

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