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More RNA extractions

Continued RNA extractions of Anthopleura elegantissima samples using the Qiagen RNeasy kit. Further information on these samples can be found in this post. Qiashredder columns were again used for sample homogenization, and the Qubit RNA HS assay for quantification. Here are the results:

Species Sample RNA (ng/ul) Total vol (ul) Notes
A. elegantissima A4-001 96 40
A. elegantissima A5-416 59 40
A. elegantissima A6-B22 >100 40
A. elegantissima H4-415 >100 40
A. elegantissima H1-518 >100 40
A. elegantissima H2-019 >100 40

RNA extraction – coral and anemone samples

I did some further RNA extractions of Porites astreoides and Anthopleura elegantissima samples using the Qiagen RNeasy kit. Further information on these samples can be found in the previous post. I again used Qiashredder columns for sample homogenization, and use the Qubit RNA HS assay for quantification. Here are the results:

Organism Sample RNA ng/ul Sample vol (ul) Notes
A. elegantissima A4 483 >100 40
A. elegantissima M4 414 <20 40
A. elegantissima H1 400 >100 40
A. elegantissima H6 120 >100 40
A. elegantissima M3 461 >100 40 lowest amount of starting material
A. elegantissima A2 438 >100 40
P. astreoides past7-cbc 75 40
P. astreoides past7-home 99 40
P. astreoides past14-cbc >100 40
P. astreoides past14-home >100 40
P. astreoides past5-cbc 67 40
P. astreoides past5-home >100 40

I may do some dilutions later.

Test RNA extraction

I just noticed that the last time I posted was about a year ago – wow, time flies! I have primarily been working on RADseq data assemblies (i.e., being a computer jockey) and have not done really any bench work since then, hence the gap.

I’m finally getting into some RNA extractions on both coral and anemone samples. The coral samples are Porites astreoides from the Belize transplant experiment, collected in November 2016. The anemone samples are Anthopleura elegantissima from Natalie Coleman’s OA experiment last summer. Both sets of samples were flash frozen in liquid nitrogen and stored in a -80°C freezer since then. In January, I crushed the majority of these samples to a fine powder under liquid nitrogen using a mortar and pestle. The coral samples ended up being more substantial and I only needed a portion of each, and the crushed powder I obtained weighed approximately 500-700 mg per sample. The anemones samples, on the other hand, were smaller, and I only got about 60-150 mg per sample. Granted, some of the weight of the coral samples is probably skeleton.

For the extractions, I used the Qiagen RNeasy kit, with initial sample homogenization with Qiashredder columns. I used the animal tissues protocol with a final elution volume of 40 µl (single pass). The following are results from the Qubit RNA HS assay:

 

Organism Sample RNA (ng/ul) Sample vol (ul) Notes
P. astreoides Past13 ’16 Home 35.9 40
P. astreoides Past13 ’16 CBC 45.7 40
A. elegantissima H2 End 422 too low 40 Low amount of starting material; thawed slightly
A. elegantissima A2 End 6 too low 40 Low-ish starting amount
A. elegantissima M3 End 468 124 60 Had to be diluted to Qubit
A. elegantissima A1 end 425 71.6 60 Had to be diluted to Qubit

 

Overall I am pleased with these results. The only one I find odd and hard to explain is H2 End 422 – this had a relatively low amount of starting material, but I didn’t think it was that low. It may have thawed or been otherwise compromised, but I made no note of this.

PBS recipe

1X Phosphate Buffered Saline (PBS Buffer) Recipe

Dissolve in 800ml distilled H2O:

8g of NaCl
0.2g of KCl
1.44g of Na2HPO4
0.24g of KH2PO4

Adjust pH to 7.4 with HCl.

Adjust volume to 1L with distilled H2O.

Sterilize by autoclaving.

Symbiodinium cp23S Re-PCR

Yesterday I completed some re-do PCRs of Symbiodinium cp23S from the branching Porites samples Sanoosh worked on over the past summer. Some of the samples did not amplify at all, so I reattempted PCR of these samples (107, 108, 112, 116). Sample 105 amplified last summer but the sequence was lousy, so I redid that one too. After the first PCR, I obtained 1 ul of the product and diluted it 1:100 in water. I then used 1 ul of this diluted product as the template for a second round of PCR. PCR conditions were the same Sanoosh and I used last summer (based on Santos et al. 2002):

Reagent Volume (µl)
water 17.2
5X Green Buffer 2.5
MgCl2 25 mM 2.5
dNTP mix 10 mM 0.6
Go Taq 5U/µl 0.2
primer 23S1 10 µM 0.5
primer 23S2 10 µM 0.5
Master Mix volume 24
sample 1
total volume 25

Initial denaturing period of 1 min at 95 °C, 35 cycles of 95 °C for 45 s, 55 °C for 45 s, and 72 °C for 1 min, and a final extension period of 7 min.

Samples were then run on a 1% agarose gel for 30 min at 135 volts.

021217_cp23s

Surprisingly, the first round of PCR amplified samples 107 and 112 (note: two subsamples of each were run; one that was the original extraction diluted (d) and another that was the original cleaned with Zymo OneStep PCR Inhibitor Removal kit (c)). The cleaned samples were the ones that amplified. I believe Sanoosh had tried these cleaned samples with no success.

The second round of PCR produced faint bands for both of the 108 samples. Sample 116 still did not amplify.

I cleaned the samples with the NEB Monarch Kit and shipped them today to Sequetech. I combined the two 108 samples to ensure enough DNA for sequencing.

RAD library prep

This is a belated post for some RAD library prep I did the week of January 23rd in the Leache Lab. I followed the same ddRAD/EpiRAD protocol I used in August. Samples included mostly Porites astreoides from the transplant experiment, as well as some geoduck samples from the OA experiment, and a handful of green and brown Anthopleura elegantissima. Sample metadata can be found here. The library prep sheet is here. The TapeStation report is here. Below is the gel image from the TapeStation report showing that the size selection was successful. However, the selection produced fragments with a mean size of 519-550 base pairs, as opposed to the size selection in August which produced ~500 bp fragments. While there will obviously be some overlap between libraries, combining samples from the two libraries may be problematic. This occurred despite identical Pippen Prep settings targeting fragments 415-515 bp. Libraries were submitted to UC Berkeley on 1/31/17 for 100 bp paired-end sequencing on the HiSeq 4000.

Library JD002_A-L

Picture1

DNA extraction: Anthopleura elegantissima

In the interest of comparing methylation levels between symbiotic states in Anthopleura elegantissima, I extracted DNA from three zooxanthellate and three zoochlorellate individuals. These were anemones that were collected last summer at Point Lawrence, Orcas Island, and had been residing in indoor sea tables at Shannon Point since then. For each specimen, I excised part of the tentacle crown with scissors and deposited the tissue directly into a microfuge tube. I opted to freeze the tissue in the -80ºC freezer since earlier attempts with fresh tissue did not seem as effective (i.e., the tissue seemed resistant to lysis). After a day or two in the freezer, I pulled the samples out, rinsed them with PBS, and proceeded with the Qiagen DNeasy assay.  After addition of proteinase K, I used a small pestle to homogenize the sample. An overnight lysis period at 56ºC was used. DNA was eluted via two passes with 50 µl AE buffer (100 µl total volume). To further clean the DNA, samples were subject to ethanol precipitation using this protocol. Samples were re-eluted in 50 µl AE buffer.

To assess DNA quantity and quality, samples were tested with the Qubit BR DNA assay followed by electrophoresis on a 1% agarose gel with 1X TBE, 135 volts for 25 min.

sample ng/µl ng/µl post-dilution (100 µl buffer) total DNA
Ae-B-1 256 128 12800
Ae-B-2 183 91.5 9150
Ae-B-3 304 152 15200
Ae-G-1 163 81.5 8150
Ae-G-2 166 83 8300
Ae-G-3 274 137 13700

Ae_DNA_011017

DNA extraction – naturally bleached Porites spp.

I was able to collect some naturally bleached Porites astreoides and Porites porites specimens while in Belize in November. I hope to use these as reference samples without symbiont DNA for RADseq. These samples were flash frozen instead of preserving in SS-DMSO. To extract DNA, a small fragment was crushed with a mortar and pestle and divided between three 1.5 ml tubes for extraction using the Qiagen DNeasy assay.  An overnight proteinase K lysis period at 56ºC was used. DNA was eluted via two passes with 50 µl AE buffer (100 µl total volume). To further clean the DNA, samples were subject to ethanol precipitation using this protocol. Samples were re-eluted in 100-200 µl AE buffer.

To assess DNA quantity and quality, samples were tested with the Qubit BR DNA assay followed by electrophoresis on a 1% agarose gel with 1X TBE, 125 volts for 30 min.

sample ng/ul (qubit) total vol total DNA ng
Past_bleached_1 15.8 100 1580
Past_bleached_2 7.02 100 702
Past_bleached_3 6.32 100 632
Ppor_bleached_1 16.7 100 1670
Ppor_bleached_2 6.74 200 1348
Ppor_bleached_3 13.7 100 1370

 

DNA looks great but note that RNase was not used and there may be some RNA in there. Also, note that the pellet was brown and there may be other contaminants.

DNA_122216

DNA extraction – transplant expt.

This week I extracted DNA from Porites astreoides samples collected the week of 11/8/16 in Belize. These samples represent the endpoint of a year long common garden experiment in the backreef at Carrie Bow Cay. Detailed sample info can be found in the initial post here. The samples were stored at room temperature in salt saturated DMSO. Tissue was scraped off the skeletons with forceps into 1.5 ml tubes. Tissue was washed three times with PBS via centrifugation before beginning the Qiagen DNeasy extraction method. An overnight proteinase K lysis period at 56ºC was used. DNA was eluted via two passes with 50 µl AE buffer (100 µl total volume). To further clean the DNA, samples were subject to ethanol precipitation using this protocol. Samples were re-eluted in 50 µl AE buffer.

To assess DNA quantity and quality, samples were tested with the Qubit BR DNA assay followed by electrophoresis of the majority of them on a 1% agarose gel with 1X TBE, 135 volts for 30 min.

Sample DNA ng/µl
19 37.6
8 302
17 256
11h 304
7 180
18 326
16 242
2 151
11 178
10 306
12 163
4h 191
5 66.8
15 50.6
94 167
14 66.8
9 91.6
3 382
1 92.2
13h 197
5h 98.4
7h 184
6 195

113016_dna

GOALS for November

November will be an important month. Here are my goals:

1. Prep for Belize trip

  1. Prepare DMSO and RNA later in 50 ml tubes
  2. Order supplies: decitabine, magnetic stirrers, heaters, etc
  3. Anything else??? Bring waterpik, hemacytometer?

2. Belize trip goals

  1. Collect transplanted P. astreoides, save separate samples for DNA and morphology
  2. Run short term (3-7 days) gene expression experiment in backreef
  3. Run two week heat stress experiment in aquaria
  4. Possibly collect more P. porites samples
  5. Consider other side projects

3. Continue analysis of RADseq data

  1. Things to consider: allow missing data? test for outlier loci? how to deal with EpiRAD data?
  2. Goal to have mostly complete analysis by the end of the month (also end of class)