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

PCR – Mac’s Bisulfite-Treated DNA

Realized that the PCR performed on 20140828 used the incorrect forward primer! As such, am repeating as before, but with the correct forward primer:

CgBS_733_26796F (SRID: 1597)

NOTE: Nothing left of sample EV2.28 bisulfite, so this was not run.

Results:

Ladder – O’GeneRuler 100bp Ladder (ThermoFisher)

Samples are loaded in numerical order from left to right, with a NTC sample before the second ladder.

All samples ran at ~275bp, which is larger than the previous gels. Confirmed with Mac that this gel looks correct. Will contact Cassie at Fred Hutchinson to go forward with PyroMark sequencing of these products.

Evidently, it would seem that Mac (and I) used the incorrect primer set when performing this PCR most recently. Doh!

PCR – Mac’s Bisulfite-Treated DNA

Per Mac’s request, ran a PCR on a set of bisulfite-treated DNA (in her gDNA 2014 box in small -20C):

  • EV2.16 bisulfite
  • EV2.20 bisulfite
  • EV2.22 bisulfite
  • EV2.24 bisulfite
  • EV2.28 bisulfite
  • EV2.29 bisulfite
  • EV2.32 bisulfite
  • EV2.33 bisulfite

DNA needed to be diluted. Diluted according to this sheet provided by Mac:

http://eagle.fish.washington.edu/bivalvia/070914bisulfite.pdf

NOTE: EV2.28 didn’t have sufficient DNA left to prepare the dilution according to Mac’s sheet. Instead, the remaining volume ofEv2.28 bisulfite DNA (0.5uL) was diluted in a total volume of 2.5uL to maintain the same dilution ratio.

Master mix calcs are here: 20140828 – PCR Mac Bisulfite Samples

Primers used were:

CgBS_733_26796Seq (SRID: 1598)
CgBS_733_26796R_5’biotin (SRID: 1596)

Cycling params:

  1. 1. 95C – 10mins
  2. 2. 94C – 30s
  3. 3. 56C – 30s
  4. 4. 72C – 30s
  5. 5. Repeat steps 2 – 5 44 more times
  6. 6. 72C – 10mins

Results:

Ladder used is O’GeneRuler 100bp DNA Ladder (ThermoFisher).

According to Mac, the expected band size is ~300bp. However, all samples are running at ~150bp. Mac is confused and does not know what to do.

*UPDATE 20140902* – Realized I used the wrong forward primer! Will repeat PCR with correct primer. Wonder if Mac did the same thing…

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).

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.

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).

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