Submitted 3μL (~75ng) of RNA from each of the two gonad samples isolated from foot tissue embedded in paraffin histology blocks 20150408 (to assess quality of RNA) to Jesse Tsai at University of Washington Department of Environmental and Occupational Health Science Functional Genomics Laboratory:
Jesse will determine if the samples should be run on the RNA Pico or the RNA Nano chips.
Ran the 400ppm library and the 1000ppm library preps on a DNA1000 Assay Chip (Agilent) on the Agilent 2100 Bioanalyzer.
Results:
Data File (XAD): 2100_expert_DNA_1000_DE72902486_2015-03-02_09-18-02.xad
Electropherogram overlay of both samples:
Red = 400ppm
Blue = 1000ppm
Measurement data and parameters are here: 20150302_Bioanalyzer_Cgigas_400_1000ppm_BS-Seq
Both libraries look good; no adaptor contamination (peak would be present at ~125bp), good library sizes.
Pooled equal quantities of each library, based off the concentration values above, to prepare the sample for sequencing.
| Component | Volume (μL) | Quantity (ng) |
| 400ppm library | 10 | 14.7 |
| 1000ppm library | 1.09 | 14.7 |
| Buffer EB | 7.81 | N/A |
| 1% Tween20 | 2.1 | N/A |
| Total | 21 | N/A |
The pooled libraries will be submitted tomorrow to the Genomics Core Facility at the Univ. of Oregon for high-throughput sequencing (100bp, SE) on the HiSeq2500 (Illumina). Sample order #2212.
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
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.
I was contacted by the sequencing facility at the University of Oregon regarding a sample quality issue with our library. As evidenced by the electropherogram below, there is a great deal of adaptor primer dimer (the peak at 128bp):
This is a problem because such a high quantity of adaptor sequence will result in the majority of reads coming off the Illumina being just adaptor sequences.
With the remainder of the library sample prepared earlier, I performed the recommended clean up procedure for removing adaptor sequences in the EpiNext Post-Bisulfite DNA Library Preparation Kit – Illumina (Epigentek). Briefly:
Added equal volume of MQ Beads
Washed beads 3x w/80% EtOH
Eluted DNA w/12uL Buffer EB (Qiagen)
After clean up, quantified the sample via fluorescence using the Quant-iT DNA BR Kit (Life Technologies/Invitrogen). Used 1uL of the sample and the standards. All standards were run in duplicate and read on a FLx800 plate reader (BioTek).
Results are here: 20150122 – LSU_virginicaMBDlibraryCleanup
Library concentration = 2.46ng/uL
Brought the entire sample up to 20uL with Buffer EB (Qiagen) and a final concentration of 0.1% Tween-20 (required by the sequencing facility).
Sent sample to the University of Oregon to replace our previous submission.
To gain a more quantitative assessment of the fragmentation from 20100625, I ran 1uL of each sample (~55ng, according to pre-fragmentation spec values) on the Agilent Bioanalyzer 2100, using the DNA 1000 kit, according to manufacturer’s protocol.
Results:
Avg. size of fragmentation is ~460bp for the two samples. Fragmentation size was determined by marking the same region on both sample’s electropherograms (see below).
R37: Avg. size = 450bp (in Region 1, marked with blue lines in image below)
R51: Avg. size = 468bp (in Region 1, marked with blue lines in image below)
Overlay of R37 and R51 fragmentation. Note that both electropherograms are nearly identical (this is good).
Amplified cDNA was cleaned up using the Invitrogen PureLink Micro Kit, but was done so according to Ambion’s Whole Transcriptome Analysis Kit protocol and then spec’d.
Results:
0.5uL was removed from each sample and mixed with 0.5uL to run on DNA 1000 chips on the Bioanalyzer 2100. The slideshow below shows the electropherograms from each sample. Each sample (to be considered worthy of moving to the next stage) should have <20% of the sample in the 25-150bp range. All 8 samples exhibit this and their peaks look very good. Will proceed to ePCR/templated bead prep next week.
0.5uL of fragmented mRNA from each library (combined with 0.5uL) was run on Agilent Bioanalyzer 2100 using RNA Pico chips/reagents according to Agilent’s protocol.
Results:
Total RNA shows a single, distinct rRNA band, along with some low-molecular weight RNA (i.e. degraded) in both total RNA samples. mRNA samples exhibit the expected “smear” that spans a large range of molecular weights. Both mRNA samples also show residual rRNA bands, but their concentrations should be extremely low. Fragmented samples show the expected strong band of low-molecular weight RNA. The CE frag sample exhibits some larger banding, which is probably background signal (compare to the empty lane labelled “Sample 7″).
Will proceed with rest of library procedure for both fragmented samples.
1uL of each sample from 20100325 was run on the Agilent 2100 Bioanalyzer on a RNA Pico 6000 chip to evaluate RNA quantity and fragmentation.
Results:
Total and mRNA aliquots (~5ng/uL) were run on the Agilent Bioanalyzer Pico RNA chips.
Results:
The gel below shows the comparison/results of total RNA and subsequent mRNA isolations. The gel indicates the following:
mRNA isolations worked for all samples. Although one might be inclined to say that mRNA isolation did NOT work for the WB sample, one has to take in to consideration that the gel software adjusts the gel contrast to enhance low signals. That’s why all the mRNA samples exhibit a dark background. mRNA generates a broad, relatively weak signal when compared to a total RNA sample. So, the software attempts to boost the low signal for display purposes. Thus, if we were to decrease this signal boosting (or contrast) for the WB mRNA so that the background color matched the WB total RNA background color (white), the rRNA bands visible in the WB mRNA sample would fade to a point where they would not be visible. See the electropherogram overlay (below the gel) for a more visual comparison of this concept.
Electropherogram Overlays of WB total RNA and WB rRNA
The WB total RNA is the red graph which shows extremely high levels of rRNA (as expected). After subsequent mRNA isolation (the blue graph), the rRNA is virtually gone and no longer comprises a significant portion of the sample.
Samples were run on the DNA 1000 chip for cDNA smear analysis.
Results: 2 of the 4 samples (2L & 3L) look perfect (<20% of cDNA in the 25-150bp range). 6L has <20% of the cDNA in the 25-150bp range (which is perfect), but exhibits a “smear” from ~250-500bp. cDNA in this range suggests overamplification, which will skew gene expression profile. Can repeat PCR for 6L using outer gel slices and reduce the number of cycles to prevent overamplification, if desired. Spoke to Steven and since these samples won’t be used to evaluate gene expression (they’re for SNP discovery), we won’t worry about it for the time being.
Sample 4L has some very strange signals being generated in the ~500-800bp range. Additionally, the virtual gel image (not shown) shows a great deal of smearing, unlike the other samples.
