Tag Archives: OA

Bisuflite NGS Library Prep – C.gigas larvae OA bisulfite library quantification

The two completed BS Illumina libraries (400ppm and 1000ppm) were quantified via fluorescence using the Quant-iT DNA BR Kit (Life Technologies/Invitrogen).  Used 1uL of  each sample and the standards.  All standards were run in triplicate.  Due to limited sample, the two libraries were only processed singularly, without replication.  Fluorescence was measured on a FLx800 plate reader (BioTek).

 

Results:

The standard curve, raw fluorescence, and calculated concentrations (as determined by the Gen5 (BioTek) software) can be seen here: 20150128_CgigasOA_BSlibrraryQuants_OluridaLibraryQuants

The standard curve was excellent, exhibiting a R² value = 0.999

 

Sample Concentration (ng/uL)
400ppm 10.592
1000ppm 0.0

 

The 400ppm library looks great, with a good yield.

The 1000ppm library appears to have no measurable quantity of DNA in it.  This is surprising, and disconcerting, as both samples were processed in parallel.  As such, there should be virtually no difference between them, in regards to the library construction process and subsequent yields.

To verify that this wasn’t a pipetting error on my part, I re-quantified the 1000ppm library (in duplicate) and still no detectable DNA.

Will repeat the bisulfite conversion and library construction process on the 1000ppm sample in order to generate a usable library for sequencing.

Bisuflite NGS Library Prep – C.gigas larvae OA bisulfite DNA

The two pooled bisulfite-treated DNA samples (400ppm and 1000ppm) from 20150114 were used to prepare bisulfite Illumina libraries with EpiNext Post-Bisulfite DNA Library Preparation Kit (Illumina) (Epigentek).  Changes to the manufacturer’s protocol:

  • Samples were transferred to 1.5mL snap cap tubes for all magnetic bead steps in order to fit in our tube magnets.
  • Stopped after End Repair step (prior to magnetic bead clean up).  Samples stored @ -20C

DNA Isolation – C.gigas larvae from 2011 NOAA OA Experiment

DNA was isolated from the following samples:

SAMPLE ID DATE TREATMENT (ppm) # LARVAE
6B5 20110513 400 5,000
1B2 20110513 1000 5,000
6B2 20110513 400 10,000
1B1 20110513 1000 10,000
1B1 20110519 1000 NA
1B2 20110519 1000 NA
6B2 20110519 400 NA
6B1 20110519 400 NA

 

Some tubes contained a high quantity of algae, based on quantity of material in tube and overall green color.

Samples 1B1 & 1B2 from 20110519 have excessive quantities of algae.

Samples 6B1 & 6B1 from 20110519 have a fair amount of algae.

See pic:

 

Sample tubes after brief spin, prior to DNA isolation.

Prior to isolation, samples were briefly spun (12,000g, 15s @ RT). Supernatants were discarded.

 

 

DNA Isolation

DNA was isolated using the DNeasy Blood & Tissue Kit (Qiagen).

Samples were resuspended in 180uL of Buffer AL and 20uL of Proteinase K. Samples were mixed by vortexing and incubated @ 56C O/N.

The manufacturer’s protocol (Purification of Total DNA from Animal Tissues (Spin-Column Protocol)) was followed.

Due to low quantities of starting tissue, samples were eluted with 200μL of Buffer EB to maximize DNA recovery.

 

DNA Quantification

Samples were prepared for quantification via fluorescence using the Quant-iT DNA BR Kit (Life Technologies/Invitrogen). The manufacturer’s protocol was altered to use 5μL of sample and 5μL of standards (instead of 10μL) in each well. All samples/standards were run in duplicate and read on a FLx800 plate reader (BioTek).

Mean fluorescence of the standards were plotted with a best-fit line. The resulting equation from the best-fit line was used to determine sample concentrations from their mean fluorescence.

 

Results:

Calcs and resulting quantities are here:

https://docs.google.com/spreadsheets/d/1e7EF05akWeBtO7Xz0UWXhIzRlkVU5HA_rjCE3c4SPEw/edit?usp=sharing

 

All samples have yields great enough to proceed with shearing and bisulfite conversion.

Samples 1B1 and 1B2 from 20110519 have extremely large yields.  This is not surprising, considering the amount of algae present in the source tubes.  Will process only 500ng from each sample.

 

 

DNA Shearing

Adjusted volume of all samples to 190μL using Buffer EB (Qiagen) in 1.5mL snap-cap tubes.

Samples were sonicated/sheared in the Bioruptor (Diagenode) with the following cycling protocol:

25 cycles of:

30s on

30s off

Cycling params were adjusted from the last time I performed this, since I felt the final sheared size was a bit on the small size.

After shearing, samples were stored @ 4C until I could SpeedVac them to reduce their volumes, as the bisulfite treatment step requires volumes < 24uL.


 

DNA Isolation – C.gigas Larvae from Emma OA Experiments

Isolated gDNA using DNazol from the following larval samples for potential MBD selection and bisulfite sequencing:

– Added 500uL of DNAzol to each tube, transferred to 1.5mL tube and homogenized with disposable pestles

– Added additional 500uL DNAzol to each homogenized sample and mixed by inversion.

– Incubated 10mins at RT

– Pelleted debris by spinning 10,000g, 10mins, @ RT

– Transferred supes to new tubes

– Added 500uL of 100% EtOH to each; mixed by inversion; incubated 10mins @ RT

– Pelleted DNA by spinning 5,000g, 4mins, @ RT

– Discarded supes

– Washed DNA with 1mL 70% DNAzol/30% EtOH solution

– Spun 1000g, 1min, @ RT

– Discard supes

– Washed DNA with 1mL 75% EtOH

– Spun 1000g, 1min, @ RT

– Discarded supes

– Spun 1000g, 1min, @ RT

– Removed residual EtOH with pipette; air dried samples for 5mins @ RT

– Added 20uL of Trish-HCl (pH = 8.0) to each sample; incubated 10mins @ RT; flicked tubes to help dissolve

– Spun 12,000g, 10mins, @ RT

– Transferred supes to new tubes

– Spec’d on NanoDrop 1000 (ThermoFisher) and recovered solution from each sample due to limited sample volume

Results:

DNA Isolation – C.gigas Larvae from Katie Latterhos

Since the previous isolation attempt was unsuccessful (see 20140922), we’re trying a slightly different approach than yesterday.

Today, I will pellet the samples, remove the RNA Later and then proceed with the Quick-gDNA MicroPrep Kit (ZymoResearch) according to the manufacturer’s protocol for Cell Suspensions and Proteinase K Digested Samples.

Isolated gDNA from two C.gigas larvae samples from Katie Latterhos:

B1 400 D6

B6 D00

Pelleted the samples at 10,000g, 5mins, RT. Although no pellets were visible in either sample, the B1 400 D6 sample did have visible cells/debris at the top of the RNA Later after spinning! So, I recovered that portion of the sample for use in the DNA isolation. The B6 D00 sample had no visible debris, nor pellets, so the RNA Later supernatant was removed and discarded.

Both samples were then processed with the Quick-gDNA MicroPrep Kit (ZymoResearch) according to the manufacturer’s protocol for Cell Suspensions and Proteinase K Digested Samples.

Samples were eluted with 10uL of elution buffer and spec’d on a NanoDrop1000 (ThermoFisher). The 2uL used for each sample were recovered from the NanoDrop.

Results:

Note: The B1 400 D6 sample was spec’d twice, due to an error message on the NanoDrop when spec’ing it the first time. Thus, the second entry for B1 400 D6 is the correct value.

Although the B1 400 D6 sample actually yielded gDNA today, the yield is far too low for use in RAD sequencing (need 500ng; B1 400 D6 yielded only ~260ng). Additionally, the quality of the DNA isolated is horrible (OD 260/280 = 0.81).

The B6 D00 did not yield any DNA.

Will let Steven know and see how he wants to proceed.

DNA Isolation – C.gigas Larvae from Katie Latterhos and Emma

Isolated gDNA from two C.gigas larvae samples (stored in RNA Later) from Katie Latterhos:

B4 400 D05

B6 400 D03

and two samples from Emma:

280E

380E

Emma’s samples were in her -80C box (in rack #2): C.gigas larvae – NOAA O.A. September 2010 Emma

Note: No visible larvae present in either of Katie Latterhos samples. Easily visible larvae in each of Emma’s samples.

DNA was isolated using the Quick-gDNA MicroPrep Kit (ZymoResearch) according to the manufacturer’s protocol for Cell Suspensions and Proteinase K Digested Samples.

Samples were eluted with 10uL of elution buffer and spec’d on a NanoDrop1000 (ThermoFisher). The 2uL used for each sample were recovered from the NanoDrop.

Results:

Not surprisingly, the samples from Katie Latterhos yielded, essentially, no gDNA. Will discuss with Steven.

*UPDATE 20141030*

Steven sent me this screen cap of Emma’s notebook so we could track where the samples originated from

qPCR – DNased Manila Clam Larvae RNA (from August 2012 – Dave’s Notebook)

Performed qPCR on Dave’s manila clam larvae DNased RNA from August 2012 using EF1a primers (SR IDs: 1463, 1474).

Master mix calcs are here. https://docs.google.com/spreadsheet/ccc?key=0AmS_90rPaQMzdHc5amwzZzdDa1d0VXQzLVU0WkFTc0E

Plate layout, cycling params, etc can be found in the qPCR Report (see Results).

Positive control was pooled cDNA taken from Dave’s cDNA plate on 8/7/2012.

Results:

qPCR Data File(CFX96) http://eagle.fish.washington.edu/Arabidopsis/qPCR/CFX96/Roberts%20Lab_2012-10-26%2010-48-07_CC009827.pcrd

qPCR Report(PDF) http://eagle.fish.washington.edu/Arabidopsis/qPCR/CFX96/Roberts%20Lab_2012-10-26%2010-48-07_CC009827.pdf

Here’s a quick Google Spreadsheet summary highlighting samples that came up positive/negative.

https://docs.google.com/spreadsheet/ccc?key=0AmS_90rPaQMzdFFHb3YwWE01UG00TnY3OWo2cWx2UVE

Approximately half of the samples (~27) came up positive for still having gDNA in them.

There are three pCO2 treatments: 1000ppm, 750ppm, and 400ppm. There are six sampling dates: 7/29/2011, 8/2/2011, 8/9/2011, 8/12/2011. Currently, it is unknown when the Day 0 samples were collected. Have emailed Dave for deets.

There are only two dates (7/29/2011 and 8/5/2011) that have a full set of samples (i.e. 1000ppm, 750ppm and 400ppm) that exhibit DNA-free RNA. Will discuss with Steven on how to proceed.

UPDATE 20121031 – Dave emailed and indicated the experimented started on 7/27/2011. Additionally, the two sample sets that are complete are Day 2 and Day 7. Discussing with Steven, we have decided to run a few genes and see how the expression levels compare to the NGS data analysis for these samples. If the qPCR data supports the NGS data, then that information will be relayed to the BMC Genomics reviewers in response to their critiques. A copy of the manuscript is here(may not be publicly viewable). https://docs.google.com/document/d/1Ii1lODz2oThiyxZtHBblUEdzyhIVq92n8jkEjhkuuts/edit