20140528
RNA-Seq - Sea Star Data Download
Received RNA-seq data from Cornell. They provided a convenient download script for retrieving all the data files at one time (a bash script containing a series of wget commands with each individual file's URL), which is faster/easier than performing individual wget commands for each individual file and faster/easier then using the Synology "Download Station" app when so many URLs are involved.
Here's the script (download.sh) that was provided:
#!/bin/bash
wget -q -c -O 3291_5903_10007_H94MGADXX_V_CF71_ATCACG_R2.fastq.gz "http://cbsuapps.tc.cornell.edu/Sequencing/showseqfile.aspx?mode=http&cntrl=1160641846&refid=17091"
wget -q -c -O 3291_5903_10007_H94MGADXX_V_CF71_ATCACG_R1.fastq.gz "http://cbsuapps.tc.cornell.edu/Sequencing/showseqfile.aspx?mode=http&cntrl=505010539&refid=17092"
wget -q -c -O 3291_5903_10008_H94MGADXX_V_CF34_CGATGT_R1.fastq.gz "http://cbsuapps.tc.cornell.edu/Sequencing/showseqfile.aspx?mode=http&cntrl=636513375&refid=17093"
wget -q -c -O 3291_5903_10008_H94MGADXX_V_CF34_CGATGT_R2.fastq.gz "http://cbsuapps.tc.cornell.edu/Sequencing/showseqfile.aspx?mode=http&cntrl=1472734408&refid=17094"
wget -q -c -O 3291_5903_10009_H94MGADXX_V_CF26_TTAGGC_R2.fastq.gz "http://cbsuapps.tc.cornell.edu/Sequencing/showseqfile.aspx?mode=http&cntrl=948605937&refid=17095"
wget -q -c -O 3291_5903_10009_H94MGADXX_V_CF26_TTAGGC_R1.fastq.gz "http://cbsuapps.tc.cornell.edu/Sequencing/showseqfile.aspx?mode=http&cntrl=1810346594&refid=17096"
wget -q -c -O 3291_5903_10010_H94MGADXX_HK_CF2_TGACCA_R2.fastq.gz "http://cbsuapps.tc.cornell.edu/Sequencing/showseqfile.aspx?mode=http&cntrl=424477466&refid=17097"
wget -q -c -O 3291_5903_10010_H94MGADXX_HK_CF2_TGACCA_R1.fastq.gz "http://cbsuapps.tc.cornell.edu/Sequencing/showseqfile.aspx?mode=http&cntrl=630586816&refid=17098"
wget -q -c -O 3291_5903_10011_H94MGADXX_HK_CF35_ACAGTG_R1.fastq.gz "http://cbsuapps.tc.cornell.edu/Sequencing/showseqfile.aspx?mode=http&cntrl=1392201335&refid=17099"
wget -q -c -O 3291_5903_10011_H94MGADXX_HK_CF35_ACAGTG_R2.fastq.gz "http://cbsuapps.tc.cornell.edu/Sequencing/showseqfile.aspx?mode=http&cntrl=1598310685&refid=17100"
wget -q -c -O 3291_5903_10012_H94MGADXX_HK_CF70_GCCAAT_R1.fastq.gz "http://cbsuapps.tc.cornell.edu/Sequencing/showseqfile.aspx?mode=http&cntrl=868072864&refid=17101"
wget -q -c -O 3291_5903_10012_H94MGADXX_HK_CF70_GCCAAT_R2.fastq.gz "http://cbsuapps.tc.cornell.edu/Sequencing/showseqfile.aspx?mode=http&cntrl=1074182214&refid=17102"
This is a bash script. However, for the most direct method of downloading these on our Synology server, we need the script to be an ash script. So, just modify the first line of the script to say "#!/bin/ash" instead of "#!/bin/bash". Then, I placed the script in the target directory for our files, SSH'd into our Synology (Eagle), changed to the directory where I placed our script (Eagle/web/whale/SeaStarRNASeq) and then ran the script (./download.sh).
20140527
RNA Isolation - Jessica's Geoduck Larval Stages
Isolated RNA from the following samples provided by Jessica Blanchette (stored in RNA later):
Trocophore 1 (T1)
Trocophore 2 (T2)
Veliger 1 (V1)
Veliger 2 (V2)
Settlers Interphase 1 (S1)
Settlers Interphase 2 (S2)
The tocophore and veliger larval stages are neutrally bouyant (i.e. will not pellet when centrifuged). In order to separate them from the RNA Later, I used a fine mesh (don't know mesh size; bag was labeled "Unknown") as a "guard" between the pipette tip and the larvae. Removed RNA Later from those two groups in this fashion. However, a significant portion of the larvae in these tubes adhered to the outside of the mesh. I left the mesh "guard" in the tube, added 1mL of TriReagent and vortexed. The mesh quickly dissolved in the TriReagent, creating a milky white mix.
For the settlers samples, there was a such a large pellet already in the existing tubes, I just took ~75uL of this material, transferred to a clean tube and added 1mL of TriReagent. However, most of the debris that I transferred dissolved extremely quickly. I was expecting there to more insoluble "debris", because marine bivalve larval shells generally don't readily dissolve in the presence of TriReagent. So, I suspect that much of the settlers samples is not really geoduck larvae.
Due to time constraints, stored all samples O/N @-80C in TriReagent.
Samples were thawed and RNA was isolated, and DNased, using the Direct-zol RNA Miniprep Kit (ZymoResearch), eluted with 50uL of 0.1% DEPC-treated H2O, and spec'd on the NanoDrop1000.
Prior to isolation, sample V1 showed a clear phase separation that none of the other samples exhibited. Sample V1 had a pink, goopy layer on top of a clear, low-viscosity layer. All other samples retained the uniform pink coloration imparted by the TriReagent. Additionally, after addition of the EtOH in the procedure to sample V1, a large amount of white precipitate formed and settled to the bottom of the tube. This did not happen in any other samples.
Samples were stored @ -80C in "
Shellfish RNA Box #5"
Results:
Overall, the yields are relatively low, as expected. Virtually all of the samples have poor OD260/280 values. Although not shown, there was a consistent shift in peak absorbance from 260nm towards 270nm, leading to the poor OD260/280 values.
20140516
DNA Isolation - Mackenzie's C.gigas EE2 Gonad Samples
Isolated DNA from the following samples, provided by Mackenzie:
EE2v2, 22.go
EE2v2, 20.go
EE2v2, 28.go
EE2v2, 29.go
EE2v2, 16.go
EE2v2, 32.go
EE2v2, 24.go
EE2v2, 33.go
Samples were suspended in 500uL of DNazol (Molecular Research Center), 5uL of PolyAcryl Carrier (Molecular Research Center), 2.75uL Proteinase K (Fermentas; 18.5mg/mL stock), briefly vortexed and incubated 24hrs at RT on rotator. Samples were briefly vortexed and insoluble material was pelleted 10,000g, 10mins, RT. Supe was transferred to fresh tube, mixed with 250uL of 100% EtOH, incubated at RT 5mins, and DNA was pelleted by spinning samples 5,000g, 5mins, RT. Supe was discarded, pellets washed with 1mL of 70% DNazol/30% EtOH solution. Supe was discarded and pellets were washed with 1mL 70% EtOH. Pellets were stored @ -20C under 95% EtOH over the weekend. Supe was discarded and pellets were washed with 70% EtOH. This step was repeated 2 more times. Supe was discarded and pellets were resuspended in Low TE Buffer, spec'd on NanoDrop1000 and run on a gel (10uL of each sample).
Results:
Yields look good and OD260/280 values look excellent. Most of the OD260/230 values aren't good, but they rarely are.
Gel Loading:
Lane 1 -
Hyperladder I (Bioline)
Lane 2 - EV2 16.go
Lane 3 - EV2 20.go
Lane 4 - EV2 22.go
Lane 5 - EV2 24.go
Lane 6 - EV2 28.go
Lane 7 - EV2 29.go
Lane 8 - EV2 32.go
Lane 9 - EV2 33.go
Lane10-
Hyperladder I (Bioline)
All samples (excluding EV2 22.go) look pretty good, with minimal smearing. All samples exhibit low molecular weight smear which is either degraded DNA or residual RNA carryover. EV2 22.go had very little tissue, so yields were expected to be extremely low. However, I was anticipating to be able to visualize it on the gel (loaded 10uL = ~90ug).
20140514
Sample Submission - Colleen Sea Star (Pycnopodia) Coelomycete RNA for Illumina Sequencing
Sent the following samples (in their entirety) to Cornell for Illumina HiSeq 100bp paired-end sequencing:
CF26 (V_CF26)
CF34 (V_CF34)
CF71 (V_CF71)
CF2 (HK_CF2)
CF35 (HK_CF35)
CF70 (HK_CF70)
20140508
RNA Isolation - Colleen Sea Star (Pycnopodia) Coelomycete Sample
Apparently the Bio26 sample provided on 20140428 was incorrect. Instead, the sample should have been CF26.
Samples were initially flash frozen and then stored @ -80C (no preservatives used). No visible cells/tissue in all samples, except Bio 26. Samples were homogenized in 1mL TriReagent. Used the Direct-zol RNA MiniPrep Kit (ZymoResearch) according to the manufacturer's protocol (including on-column DNase I procedure) for the remainder of the isolation. Eluted with 50uL of 0.1%DEPC-treated H2O and spec'd on NanoDrop1000.
Samples were stored in Shellfish RNA Box #5.
Results:
Yield and quality look great. Will pass info on to Steven and Colleen for decision on which samples to sequence.
*UPDATE 20140514* - Sample sent to Cornell for Illumina RNA-seq on 20140514
20140428
RNA Isolation - Colleen Sea Star (Pycnopodia) Coelomycete Samples
Isolated RNA from the following samples (provided by Colleen Burge):
Bio 26 (a LARGE amount of tissue/debris in this sample!)
CF 2
CF 3
CF 17
CF 34
CF 35
CF 70
CF 71
Samples were initially flash frozen and then stored @ -80C (no preservatives used). No visible cells/tissue in all samples, except Bio 26. Samples were homogenized in 1mL TriReagent. Used the Direct-zol RNA MiniPrep Kit (ZymoResearch) according to the manufacturer's protocol (including on-column DNase I procedure) for the remainder of the isolation. Eluted with 50uL of 0.1%DEPC-treated H2O and spec'd on NanoDrop1000.
Samples were stored in
Shellfish RNA Box #5.
Results:
Samples CF 3 and CF 17 likely have insufficient total RNA for sequencing at Cornell (200ng minimum required).
*UPDATE 20140514* - CF2, CF34, CF35, CF70, CF71 sent to Cornell for Illumina RNA-seq on 20140514
20140425
DNA Isolation - Claire's C.gigas Female Gonad
Trying this sample again(!!), but will now use TE for pellet resuspension to prevent sample degradation. Incubated sample RT on rotator in 500uL of DNazol + 2.7uL of Proteinase K (Fermentas; Stock 18.5mg/mL) for 5hrs. Added additional 500uL of DNazol, mixed gently and followed DNazol manufacturer's protocol. Performed first pellet was with 70% DNazol/ 30% EtOH solution. Resuspended pellet in 200uL of TE and spec'd on NanoDrop1000.
Results:
Yield is good. 260/280 value is good. 260/230 value is poor. Will run on gel to evaluate integrity.
Loaded 10uL (~830ng) on 1.0% agarose 1x modified TAE gel stained with EtBr.

Gel Loading Guide:
Lane 1 - Hyperladder I (Bioline)
Lane 2 - C.gigas female gonad gDNA (CgF)
Well, this certainly looks much better than previous preparations, in that there is an obvious high molecular weight band present (previously, this had been absent). The low molecular weight bands/smears are possibly RNA carryover and/or degraded DNA. Will discuss with Steven and then, most likely, bring downtown for Illumina sequencing.
UPDATE 20140508: Downtown sequencing facility says there's only ~800ng of DNA! This is a far cry from the minimum amount needed for sequencing (6ug). Looking at the gel above and comparing sample band intensity to the ladder band intensities suggests that the downtown sequencing facility is correct. I loaded 10uL of DNA on the gel and the intensity of the high molecular weight band is similar to the 400bp band intensity. This corresponds to 40ng of DNA. That means the CgF gDNA band is 40ng/10uL = 4ng/uL. I resuspended the gDNA pellet in 200uL of TE, so 200uL x 4ng/uL = 800ng; exactly what the sequencing facility says they measured...
I'm not entirely sure what is happening here. Until very recently, there were almost never such egregious differences between the NanoDrop measurements and what they were measuring downtown at the sequencing facility. It seems as though they have changed the way they quantify samples (possibly using an Agilent Bioanalyzer instead of the Life Technologies Qubit fluorometer?), but this doesn't mean their measurements are incorrect. However, I'm starting to suspect that the reason the initial sequencing of this sample was due to an overestimation of the quantity of input DNA (since I believe they were still using the fluorometer back then).
As such, it's become clear that C.gigas gonad samples seem to yield poor quantities of gDNA, relative to the amount of input material. Additionally, there may be insufficient sample left to generate a useable quantity of gDNA to complete this resequencing effort.
Ethanol Precipitation - Colleen's Sea Star Coelomycete RNA from Yesterday
Performed an EtOH precipitation on the sea start RNA due to some residual column resin (?) in the tubes after elution.
Added 0.1 volumes of 3M sodium acetate (pH=5.2; 10uL), 1uL glycogen (Ambion stock 5mg/mL), 2.5 volumes of ice cold 100% EtOH (275uL). Vortexed and incubated O/N at -20C.
Pelleted RNA 16,000g, 30mins @ 4C.
Discarded supe.
Washed pellet 70% EtOH.
Pelleted RNA 16,000g, 15mins @ 4C.
Repeated wash and spin.
Removed supe, resuspended RNA in 50uL of 0.1%DEPC-treated H2O and spec'd on NanoDrop1000.
Samples were stored in
Shellfish RNA Box #5.
Results:
Well, overall, the RNA looks immensely better than yesterday. However, as expected, there has been some slight loss with all the additional manipulations. As such, yields are low (although, they were initially low, too). However, I think most of the samples will be useable, albeit bordering on the minimum amount of total RNA needed (200ng) at the Cornell sequencing facility...
20140424
RNA Clean Up - Colleen's Sea Star Coelomycete RNA from 20140416
Zymoresearch support suggested putting the samples through another set of columns to help clean up the apparent phenol carryover that was seen (absorbance peak shifted to 270nm) in the initial isolation of these samples.
Added 500uL of TriReagent to each sample and vortexed. Then, proceeded with the remainder of the protocol (excluding the DNase step). Eluted with 50uL of 0.1% DEPC-treated H2O and spec'd on NanoDrop1000.
Results:
Absolutely horrible!! I can't even begin to fathom what has happened here. The samples run with the sample kit all worked so well; why did this whole thing have to be jacked up with the actual samples??!!
Well, I'll do a second elution using 50uL of 0.1%DEPC-treated H2O and spec. Let's see if that helps....
OK, I didn't even bother specing all the samples because I noticed that the elution tubes had pellets in them! When I mix the tube prior to specing (which is my normal behavior), I get the top absorbance spectra that is virtually useless. When I don't mix the samples (thus, not disturbing the pellet), I get a more "realistic" spectra, but I can't tell if I can trust it or not. I have contacted Zymoresearch support for more help with this...
It's tempting to simply proceed with an EtOH precipitation, but I'm a bit concerned that the pellet in the tubes is resin from the column and that it might still bind some of the RNA. However, I guess the pellet is already in the elution solution, so the RNA should be soluble and, theoretically, not be able to bind to any residual resin...
20140416
RNA Isolation - Colleens' Sea Star Coelomycetes Samples
Isolated RNA from the following samples stored in RNAlater:
TH52 3.28.14 c-fluid
TH54 3.28.14 c-fluid
CH55 3.28.14 c-fluid
CH56 3.28.14 c-fluid
CH57 3.28.14 c-fluid
TH65 3.28.14 c-fluid
TH66 3.28.14 c-fluid
TH67 3.28.14 c-fluid
Spun samples 5000g, 20mins @ RT to pellet any cells. Discarded supe. Resuspended cells/debris in 1mL TriReagent. Disrupted cells by pipetting and vortexting. RNA was isolated using the Direct-zol RNA Miniprep Kit (ZymoResearch). RNA was DNase treated on-column, as described in the manufacturer's protocol, using DNase I. RNA was eluted from the columns using 25uL of nuclease-free H2O and spec'd on a NanoDrop1000.
Results:
So, this is disheartening. Overall, the RNA looks pretty crappy; poor 260/280 ratios and a general shift in absorbance to 270nm. Plus, the yields aren't that great. Maybe RNA left on the column and/or some sort of contaminant pushing these readings out of whack?
I will perform another elution on the columns with 50uL of nuclease-free H2O and spec that elution set:
There's still a shift in the peak absorbance in most samples to 270nm... I'm going to combine the two sets of elutions and spec:
Although the 260/280 values are significantly better, there's still this persistent shift of peak absorbance to 270nm. I contacted technical support for the kit and they say the absorbance shift is indicative of phenol contamination. They have advised that I add a volume of TriReagent to the RNA and re-run it through a new set of columns, following the entire RNA isolation protocol.
DNA Isolation - Test Sample
Due to the recent poor quality gDNA that has been isolated from C.gigas gonad, I decided to do a quick test using TE for DNA pellet resuspension in hopes that old Buffer EB (Qiagen) or old nuclease-free H2O (Promega) are to blame for the apparent, rapid degradation that I've experienced.
Isolated gDNA from a C.gigas female gonad sample (EV2 141 go) provided by Mac. Isolated gDNA using DNazol (Molecular Research Center):
1. Incubated ~25mg of tissue O/N @ RT in 500uL of DNazol + 100ug/mL Proteniase K (2.7uL of 18.5mg/mL Fermentas stock) on rotator.
2. Added additional 500uL of DNazol and briefly disrupted remaining tissue with a few pipette strokes.
3. Pelleted debris by spinning 10mins, 10,000g @ RT.
4. Transferred supe to new tube and repeated Steps 3 & 4 one time.
5. Added 500uL of 100% EtOH; mixed by inversion.
NOTE: Despite initial appearance of white cloudy appearance after EtOH addition, cloudiness dispersed upon inversion and no visible DNA strands were present
6. Pelleted DNA by spinning 5000g 5mins @ RT.
7. Removed supe and washed pellet with 1mL of a 70% DNazol+30% EtOH solution.
8. Removed supe and washed pellet with 1mL 70% EtOH.
9. Repeated Step 8 two times.
10. Discarded supe, quick spun tube to pool residual EtOH. Removed all residual EtOH.
11. Resuspended in 200uL of TE (pH = 8.0) and incubated at RT for 5mins.
12. Pelleted insoluble material 12,000g 10mins @ RT.
13. Transferred supe to clean tube.
14. Spec'd on NanoDrop1000.
15. Ran ~500ng on 1.0% agaroase 1x modified TAE gel to evaluate integrity.
Results:
260/280 value looks excellent, but, as always seems to be the case with DNazol/TriReagent, the 260/230 value looks crappy. Will investigate gDNA integrity on agarose gel.
Gel Loading:
Lane 1 - Hyperladder I (Bioline)
Lane 2 - EV2 141 go C.gigas female gonad gDNA
Well, look at that! A nice, clear, high molecular weight band! It looks like my Buffer EB and/or nuclease-free water are is contaminated. Have discarded both. Will re-isolated Claire and Mac's gDNA.
20140414
Phenol-Chloroform DNA Clean Up - Mac and Claire's Samples (from 20140410)
Due to low 260/230 values and Mac's smeary sample, performed a phenol-chloroform DNA cleanup on the samples isolated 20140410.
1. Brought volume of each sample to 200uL with Buffer EB (Qiagen).
2. Added an equal volume (200uL) of 25:24:1 Phenol/Chloroform:Isoamyl alcohol.
3. Mixed on rotator for 20mins @ RT.
4. Separated aqueous/organic phases by spinning at 12,000g 5mins @ RT.
5. Transferred aqueous phase to new tube. Repeated steps 2-4 until samples exhibited no more interphase. Combined aqueous phases in to a single tube for each of the two samples.
6. Added and equal volume of chloroform (170uL).
7. Mixed on rotator for 20mins @ RT.
8. Separated aqueous/organic phases by spinning at 12,000g 5mins @ RT.
9. Transferred aqueous phase to new tube.
Performed an ethanol precipitation on each sample.
1. Added 0.1 volumes of 5M sodium acetate (pH = 5.2).
2. Added 2 volumes of ice cold 100% EtOH.
3. Incubated 20mins @ -20C.
4. Pelleted DNA by spinning 16,000g, 20mins @ 4C.
5. Discarded supe and washed pellets with 1mL 70% EtOH.
6. Pelleted DNA by spinning 16,000g, 5mins @ 4C.
7. Repeated steps 5-6 one time.
8. Removed all supernatant and resuspended in 100uL of nuclease-free H2O.
9. Spec'd on NanoDrop1000.
NOTE: Mac's sample exhibited the same chunky/cloudiness upon addition of 100% EtOH that has been seen previously by both her and myself...
Results:
So, the clean up seemed to work wonders on the 260/230 values. Not surprisingly, Mac's sample didn't clean up nearly as nicely as Claire's, based on my observations of the odd behavior during EtOH precipitation.
And, despite the nice, clean looking peaks, the 260/280 ratios are actually WORSE than the original isolation. Will run on gel for a further assessment of quality/integrity.
Loaded 5uL of each sample (~600ng) on a 1.0% agarose, 1x modified TAE gel stained with ethidium bromide.
Gel Layout:
Lane 1 - Hyperladder I (Bioline)
Lane 2 - Claire's CgF gonad sample
Lane 3 - Mac's gonad sample
Used Hyperladder I this time, which has a high molecular weight band of 10kb and a low molecular weight band of 200bp.
Well, this totally sucks. Both samples appear to consist of nothing but 150-200bp fragments. Is something actually degrading these samples? The Buffer EB I used during the initial extraction is certainly old. Possible source of degradation? Ugh. Maybe I'll try this again, but resuspend in TE...
20140410
RNA Isolation - Sea Star Coelomocytes (from Colleen)
Isolated RNA from two samples stored in RNAlater that had either no visible pellet or a minutely visible pellet:
Control P26
Filt. Inj. P8
Samples were spun 5000g, 20mins @ RT. Supe was removed, being sure to leave behind any debris that failed to pellet. Samples were homogenized in 1mL of TriReagent by pipetting/vortexing. RNA was then isolated using the Direct-zol RNA Miniprep Kit (ZymoResearch). RNA was eluted from the column with 25uL of 0.1%DEPC-treated H2O and spec'd on a NanoDrop1000.
Results:
RNA quality looks very good, as do the yields. I'm very surprised I got anything close to 1ug out of either sample!
However, it should be noted that neither of these samples has been DNased and, as such, the yields seen above may potentially include residual gDNA carryover which would artificially inflate the yields seen above. Will DNase the samples to see how yields are affected (if at all).
DNA Isolation - Claire's C.gigas Female Gonad and Mac's C.gigas Gonad
Due to the poor quality DNA yielded by the DNeasy Kit (Qiagen; see 20140404), I am re-isolating these samples using DNazol (Molecular Research Center). Weighed tissue from each frozen sample:
Claire's (Female DNA; 5/6/2013) - 0.022g
Mac's (EV2 9.g) - 0.017g
Incubated samples in 500uL of DNazol + 100ug/mL Proteinase K (2.7uL of 18.5mg/mL stock) O/N at RT on rotator. An additional 500uL of DNazol was added, mixed by pipetting to break up remaining tissues clumps. Manufacturer's protocol was followed, substituting the first EtOH wash with a wash of 70% DNazol, 30% 100% EtOH. Samples were resuspended in 100uL Buffer EB (Qiagen) and spec'd on a NanoDrop1000.
NOTE: Mac's sample seemed to get "chunky"/cloudy during the precipitation portion of the procedure. Claire's remained clear. Although not noted, Mac's sample behaved in a similar fashion when adding Buffer AL to the sample when using the Qiagen DNeasy Blood & Tissue Kit. Finally, Mac has previously mentioned this behavior to me as well.
Results:
Suprisingly high yields from Mac's sample.
Both samples exhibit poor 260/230 ratios and high absorbance at 230nm is evident in both samples. Mac's sample may benefit from
Ran ~600ng of each sample on a 0.8% 1x modified TAE agarose gel to visually assess sample quality.
Gel Loading (from left to right):
1. Hyperladder II (Bioline)
2. Claire's Female DNA
3. Mac's gonad (EV2 9.go)
I knew the ladder was of little use due to high molecular weight of gDNA, but it still serves as a bit of a reference. Highest molecular weight band is 2000bp.
Claire's sample looks pretty good, in relation to the lack of smearing. A single, high molecular weight band is present (albeit, faint) with almost no smearing. However, I'm disappointed by the lack of definition in the band. I fully expected a sharper, more defined band.
Mac's sample shows a high molecular weight band and significant smearing. Smearing could be indicative of either DNA degradation or high amounts of RNA carryover. If the latter, could explain the high yield.
Will attempt to clean up both samples (RNase and/or do a chloroform clean up).
20140404
Cloned Hard Drive - Windows XP Opticon Computer (Aquacul8)
Clone the XP hard drive of the computer hooked up to the Opticon qPCR machine using Macrium Reflect Free. Verified that the clone is bootable and operates correctly. Will store one of the hard drives.
DNA Gel - Claire's C.gigas Female Gonad and Mac's C.gigas Gonad
Ran out 2uL of Clair'es C.gigas female gonad gDNA (from 20140328) and Mac's C.gigas gonad gDNA (from 20140402) for quality assessment. Both samples had been isolated using Qiagen's Blood & Tissue DNeasy Kit. 2uL of each sample was run on a 0.8% 1x TBE gel.
Results:
Loading:
Lane 1 - Hyperladder 1 (Bioline)
Lane 2 - Claire's gDNA
Lane 3 - mac's gDNA
Both samples show an extremely high amount of smearing. Additionally, both samples have definitive bands that correspond to ~1300bp and ~850bp.
20140402
RNA Isolation - Sea Star Coelomocytes (provided by Colleen Burge)
Tried another method of RNA Isolation for comparison with regular TriReagent method.
Used the Direct-zol RNA MiniPrep Kit (Zymo Research) on the following samples stored in RNAlater:
P6 Control
P16 Filt. Inj.
Pelleted samples in RNAlater by spinning 5000g, 10mins @ RT. Removed RNAlater, lysed pellets in 1mL TriReagent. Split each sample equally into two tubes (500uL in each tube). Added equal volumes of 100% ethanol to each tube and vortexed. Transferred samples to spin columns and followe manufacturer's protocol. Eluted with 25uL of nuclease-free H2O (provided in kit). Spec'd on NanoDrop1000.
Results:
RNA quality is very good (based on 260/280 ratios). This turned out much better than the previous attmpt using the basic TriReagent method. However, the previous attempt (see 20140401) may have been compromised by me being too aggressive when collecting the aqueous phase. Knowing how little sample was present, I may have been overzealous in trying to gather too much of the aqueous phase, leading to the phenol carryover that was evident.
Regardless, these columns seem to do an excellent job of eliminating even salt carryover, as we frequently see high absorbance at 230nm with marine samples; particularly those stored in RNAlater.
DNA Isolation - Mackenzie's C.gigas Gonad Sample
Mac's been having some difficulty getting good quality gDNA from some of her gonad samples, so she asked me to give it a shot.
Isolated DNA from 10mg (0.010g) of C.gigas gonad tissue using the Qiagen Blood & Tissue DNeasy Kit, with the following changes:
1. Incubated sample in Buffer ATL + Proteinase K @ 56C for 3hrs
2. Eluted sample in 100uL of Buffer AE.
Spec'd on NanoDrop1000.
Results:
DNA looks good, both 260/280 ratio and yield. The 260/230 ratio isn't perfect, but it's much better than what Mac was seeing. After showing her this, she's decided to have me isolate DNA from the rest of her samples.
20140401
RNA Isolation - Sea Star Coelomocytes (provided by Colleen Burge)
Isolated RNA from the following samples (stored in RNAlater):
P18 Control 3/17/14
P10 Filt. Inj. 3/17/14
These were "trial" RNA isolation runs to determine what yields we could expect from samples of this nature.
Both samples had very small tissue/cell pellets. Tubes were spun @ 5000g for 10mins at RT to ensure all cells were pelleted. RNAlater was removed and pellets were lysed using 1000uL of TriReagent, supplemented with 8uL of PolyAcryl carrier. PolyAcryl Carrier was used to enhance RNA recovery from such small starting materials. Remainder of procedure followed manufacturer's protocol. RNA was resuspended in 20uL of 0.1% DEPC-H2O and spec'd on a NandoDrop1000.
Results:
As can be seen by the absorbance spectrum plots (top image), there is clear phenol contamination (indicated by shift of absorbance peak to 270nm, instead of the peak being at 260nm). Additionally, there're large peaks at 230nm in each of the two samples, suggesting other contamination (high residual salts, ethanol?). Additionally, the 260/280 ratios are subpar for RNA quality (i.e. <1.9). However, these ratios could be skewed by the the residul phenol present in both samples. I may perform an ethanol precipitation on these just to see if I can get them cleaned up.
Yields for both samples are very promising.
20140328
DNA Isolation - C.gigas Female Gonads (from frozen)
Isolated gDNA from Claire's "Female DNA" (from 05/16/2013) using the Qiagen Blood & Tissue DNeasy Kit according to the manufacturer's protocol, with the following changes:
1. Incubated sample in Buffer ATL + Proteinase K @ 56C for 3hrs. Vortexed once each hour.
2. Eluted with 100uL of Buffer AE.
Results:
Excellent yield and quality is good, although both the 260/280 and 260/230 ratios are on the high side. However, these high values could be an artifact of the high sample concentration (this is a common "issue" with the NanoDrop).
20140314
DNA Quality Check - Yanouk's Oyster gDNA
We've had some Illumina sequencing issues with Yanouk's samples, so I ran the samples out on a 0.8% agarose gel to evaluate the levels of degradation. Loaded 2uL of each sample. Did not load equal quantities of gDNA, due to the lack of available gDNA in the samples we submitted for Illumina sequencing. Added 2uL of H2O to samples 37 & 38 in hopes of having sufficient DNA for visualization on the gel.
See
Sam's Notebook 20131004 for sample concentrations.
Results:
First lane is Hyperladder II (Bioline). Highest molecular weight of the ladder is 2kb.
Sample numbers are listed above each lane.
All samples show a significant amount of smearing, but all still have an identifiable high molecular weight band. Will show to Steven and discuss options for re-sequencing.
20140219
DNA Isolation - Geoduck
Isolated additional geoduck gDNA from the two fresh (now frozen) geoduck's that Brent provided me with on 20140212 so that we can potentially isolate RNA from the same geoducks to tie in with the DNA Illumina sequencing that Axa will be conducting. Isolated DNA using the DNeasy Blood & Tissue Kit (Qiagen) according to the manufacturer's protocol (incubated minced siphon tissue at 56C for 3hrs). Eluted with 75uL of ddH2O and spec'd on NanoDrop1000.
Note: Initial specs were too low for Axa's requirements (50uL, >= 500ng/uL). SpeedVac'd samples to concentrate, brought volume to 55uL and then spec'd on NanoDrop1000.
Results:
Samples look good. Will send Axa 50uL of all samples, excluding GD01 since that sample is below his desired concentration AND I believe he probably doesn't want to wait for this DNA any longer.
20140218
DNA Precipitation - Geoduck DNA from 20140213
After speaking with Axa regarding the DNA concentrations, he would like the DNA from the ethanol-fixed tissue to be more concentrated, and he wants them in ddH2O instead of Buffer AE (from the Qiagen DNeasy Kit). So, I preformed a standard ethanol precipitation. Added 0.1 volumes of 3M sodium acetate (pH = 5.2) [15uL], 2.5 volumes of 100% ethanol [412.5uL] and incubated @ -20C over the weekend.
Pelleted DNA by spinning 16,000g, 15mins @ 4C. Discarded supe, washed pellets with 1mL 70% ethanol and re-pelleted the DNA. Performed a second wash with 70% EtOH, pelleted DNA, discarded supe, resuspended DNA in 75uL of NanoPure H2O, and spec'd on NanoDrop1000.
Results:
Will spec when I re-isolate DNA from "fresh" geoduck samples for coordinating Axa's DNA sequencing project with our potential RNA-seq project. See 20140219.
20140213
DNA Isolation - Geoduck
Since yesterday's DNA isolation failed to yield sufficient quantity of DNA from the ethanol-fixed samples, I isolated additional DNA from the same samples.
Isolated gDNA from 6 geoduck siphon samples provided by Brent using the Qiagen DNeasy Spin Kit. Samples were as follows:
Langley 006
Langley 007
Langley 008
Langley 009
The Langley samples were fixed in ethanol in 2006. Geoduck 01/02 were fresh geoduck siphon samples, taken from two live, juvenile geoduck (the rest of the bodies were frozen at -80C).
The samples were processed according to the Qiagen protocol (but utilized an overnight incubation at 56C with Proteinase K), eluted in 50uL of Buffer AE, combined with the corresponding DNA from yesterday, mixed throughly and spec'd on a NanoDrop1000.
Results:
Now have sufficient quantity of DNA for all four of these samples. Will contact Axa (the person who this DNA is intended for) to see if he requires a specific concentration/volume.
20140212
DNA Isolation - Geoduck
Isolated gDNA from 6 geoduck siphon samples provided by Brent using the Qiagen DNeasy Spin Kit. Samples were as follows:
Langley 006
Langley 007
Langley 008
Langley 009
Geoduck 01
Geoduck 02
The Langley samples were fixed in ethanol in 2006. Geoduck 01/02 were fresh geoduck siphon samples, taken from two live, juvenile geoduck (the rest of the bodies were frozen at -80C).
The samples were processed according to the Qiagen protocol (but utilized an overnight incubation at 56C with Proteinase K), eluted in 100uL of Buffer AE and spec'd on a NanoDrop1000.
Results:
The person who needs these samples (Axa) needs at least 25ug of DNA. The two fresh samples (Geoduck 01 and Geoduck 02) yielded more than sufficient quantities of DNA. The Langley (i.e. ethanol-fixed) samples did not yield sufficient DNA and I will need to isolate additional DNA from these samples.