Category Archives: Protein expression profiles during sexual maturation in Geoduck

SRA Release – Transcriptomic Profiles of Adult Female & Male Gonads in Panopea generosa (Pacific geoduck)

The RNAseq data that I previously submitted to NCBI short read archive (SRA) has been released to the public today. Here are the various links for the project:

Study: SRP072283 – http://trace.ncbi.nlm.nih.gov/Traces/study/?acc=SRP072283

 

BioProject: PRJNA316216 – http://www.ncbi.nlm.nih.gov/bioproject/PRJNA316216

Study: SRP072283 – http://trace.ncbi.nlm.nih.gov/Traces/sra/?study=SRP072283

Female Pool Experiment: SRX1659865 – http://www.ncbi.nlm.nih.gov/sra/SRX1659865

Male Pool Experiment: SRX1659865 – http://www.ncbi.nlm.nih.gov/sra/SRX1659866

SRA Submission – Transcriptomic Profiles of Adult Female & Male Gonads in Panopea generosa (Pacific geoduck).

RNAseq experiment, which is part of a larger project that involves characterizing geoduck gonad development across multiple stages: histologically, proteomically, and transcriptomically. Initial sample collection performed by Grace Crandall.

The current status can be seen in the screen cap below. Current release date is set for a year from now, but will likely bump it up. Need Steven to review the details of the submission (BioProject, Experiment descriptions, etc.) before I initiate the public release. Will update this post with the SRA number once we receive it.

Here’s the list of files uploaded to the SRA:

Geo_Pool_F_GGCTAC_L006_R1_001.fastq.gz
Geo_Pool_F_GGCTAC_L006_R2_001.fastq.gz
Geo_Pool_M_CTTGTA_L006_R1_001.fastq.gz
Geo_Pool_M_CTTGTA_L006_R2_001.fastq.gz

Mate pair sequencing files were uploaded together within a single “Run”.

RNAseq Data Receipt – Geoduck Gonad RNA 100bp PE Illumina

Received notification that the samples sent on 20150601 for RNAseq were completed.

Downloaded the following files from the GENEWIZ servers using FileZilla FTP and stored them on our server (owl/web/nightingales/P_generosa):

Geo_Pool_F_GGCTAC_L006_R1_001.fastq.gz
Geo_Pool_F_GGCTAC_L006_R2_001.fastq.gz
Geo_Pool_M_CTTGTA_L006_R1_001.fastq.gz
Geo_Pool_M_CTTGTA_L006_R2_001.fastq.gz

Generated md5 checksums for each file:

$for i in *; do md5 $i >> checksums.md5; done

Made a readme.md file for the directory.

Sample Submission – Geoduck Gonad for RNA-seq

Prepared two pools of geoduck RNA for RNA-seq (Illumina HiSeq2500, 100bp, PE) with GENEWIZ, Inc.

I pooled a set of female and a set of male RNAs that had been selected by Steven based on the Bioanalyzer results from Friday.

The female RNA pool used 210ng of each sample, with the exception being sample #08. This sample used 630ng. The reason for this was due to the fact that there weren’t any other female samples to use from this developmental time point. The two other developmental time points each had three samples contributing to the pool. So, three times the quantity of the other individual samples was used to help equalize the time point contribution to the pooled sample. Additionally, 630ng used the entirety of sample #08.

The male RNA pool used 315ng of each sample. This number differs from the 210ng used for the female RNAs so that the two pools would end up with the same total quantity of RNA. However, now that I’ve typed this, this doesn’t matter since the libraries will be equalized before being run on the Illumina HiSeq2500. Oh well. As long as each sample in each pool contributed to the total amount of RNA, then it’s all good.

The two pools were shipped O/N on dry ice.

  • Geo_pool_M
  • Geo_pool_F

Calculations (Google Sheet): 20150601_Geoduck_GENEWIZ_calcs

Bioanalyzer – Geoduck Gonad RNA Quality Assessment

Before proceeding with transcriptomics for this project, we need to assess the integrity of the RNA via Bioanalyzer.

RNA that was previously isolated on 20150508, 20150505, 20150427, and 20150424 (those notebook entries have been updated to report this consolidation and have a link to this notebook entry) were consolidated into single samples (if there had been multiple isolations of the same sample) and spec’d on the Roberts Lab NanoDrop1000:

Google Sheet: 20150528_geoduck_histo_RNA_ODs

NOTE: Screwed up consolidation of Geoduck Block 03 sample (added one of the 04 dupes to the tube, so discarded 03).

RNA was stored in Shellfish RNA Box #5.

RNA was submitted to to Jesse Tsai at University of Washington Department of Environmental and Occupational Health Science Functional Genomics Laboratory for running on the Agilent Bioanalyzer 2100, using either the RNA Pico or RNA Nano chips, depending on RNA concentration (Pico for lower concentrations and Nano for higher concentrations – left decision up to Jesse).

 

Results:

Bioanalzyer 2100 Pico Data File (XAD): SamWhite_Eukaryote Total RNA Pico_2015-05-28_12-50-00.xad
Bioanalzyer 2100 Nano Data File (XAD): SamWhite_Eukaryote Total RNA Nano_2015-05-28_13-22-53.xad

 

Pico Gel Representation

 

Pico Electropherogram

 

Nano Gel Representation

 

Nano Electropherogram

 

Jesse alerted me to the fact that they did not have any ladder to use on the Nano chip, as someone had used the remainder, but failed to order more. I OK’d him to go ahead with the Nano chip despite lacking ladder, as we primarily needed to assess RNA integrity.

 

Bad Samples:

  • Geo 04 – No RNA detected
  • Geo 65, 67, 68 – These three samples show complete degradation of the RNA (i.e. no ribosomal band present, significant smearing on the gel representation).

All other samples look solid. Will discuss with Steven and Brent on how they want to proceed.

Full list of samples for this project (including the Block 03 sample not included in this analysis; see above). Grace’s notebook will have details on what the numbering indicates (e.g. developmental stage).

  • block 02
  • block 03 (no RNA)
  • block 04 (no RNA)
  • block 07
  • block 08
  • block 09
  • block 34
  • block 35
  • block 38
  • block 41
  • block 42
  • block 46
  • block 51
  • block 65 (degraded RNA)
  • block 67 (degraded RNA)
  • block 68 (degraded RNA)
  • block 69
  • block 70

RNA Isolation – Geoduck Gonad in Paraffin Histology Blocks

UPDATE 20150528: The RNA isolated in this notebook entry may have been consolidated on 20150528.

The RNA isolation I performed earlier this week proved to be better for some of the samples (scraping tissue directly from the blocks), but still exhibited low yields from some samples. I will perform a final RNA isolation attempt (the kit only has six columns left) from the following samples:

  • 02
  • 03
  • 04
  • 07
  • 08
  • 09

Instead of full sections from each histology cassette, I gouged samples directly from the tissue in each of the blocks to maximize the amount of tissue input.

IMPORTANT:

Samples were then processed with the PAXgene Tissue RNA Kit in a single group.

Isolated RNA according to the PAXgene Tissue RNA Kit protocol with the following alterations:

  • “Max speed” spins were performed at 19,000g.
  • Tissue disruption was performed with the Disruptor Genie @ 45C for 15mins.
  • Shaking incubation step was performed with Disruptor Genie
  • Samples were eluted with 40μL of Buffer TR4, incubated @ 65C for 5mins, immediately placed on ice and quantified on the Roberts Lab NanoDrop1000.

 

All samples were stored @ -80C in Shellfish RNA Box #5.

Results:

 

Two samples (02 and 07) produced great yields and perfect RNA (260/280 and 260/230 of ~2.0). The remainder of the samples showed little improvement compared to what I’ve been obtaining from the previous three attempts. Will discuss with Steven and Brent about how to proceed with this project.

RNA Isolation – Geoduck Gonad in Paraffin Histology Blocks

UPDATE 20150528: The RNA isolated in this notebook entry may have been consolidated on 20150528.

Last week’s RNA isolation (a second attempt at obtaining RNA from the samples) performed poorly. I will re-isolate RNA from the following samples:

  • 02
  • 03
  • 04
  • 07
  • 08
  • 09
  • 35
  • 38
  • 46
  • 65
  • 67
  • 68

Instead of full sections from each histology cassette, I gouged/shaved off samples directly from the tissue in each of the blocks to maximize the amount of tissue input. However, due to the small size and susceptibility to flying around because of static electricity, none of these were able to be weighed prior to processing.

IMPORTANT:

Samples were then processed with the PAXgene Tissue RNA Kit in a single group.

Isolated RNA according to the PAXgene Tissue RNA Kit protocol with the following alterations:

  • “Max speed” spins were performed at 19,000g.
  • Tissue disruption was performed with the Disruptor Genie @ 45C for 15mins.
  • Shaking incubation step was performed with Disruptor Genie
  • Samples were eluted with 40μL of Buffer TR4, incubated @ 65C for 5mins, immediately placed on ice and quantified on the Roberts Lab NanoDrop1000.

Results:

 

 

 

Well, despite the low numbers, all of the samples (excluding 46 – 68) are double the yield of what I saw previously. This is good, but the amount of RNA from these is probably borderline sufficient quantity for RNA-Seq.

The kit has enough columns for six sample preps. I think I’ll attempt this strategy again (gouging/shaving directly from tissue in histo cassette), but really take a fair amount of tissue this time and see if I can get more.

All samples were stored @ -80C in Shellfish RNA Box #5.

Goals – May 2015

Here are the things I plan to tackle throughout the month of May:

Geoduck Reproductive Development Transcriptomics

My primary goal for this project is to successfully isolate RNA from the remaining, troublesome paraffin blocks that have yet to yield any usable RNA. The next approach to obtain usable quantities of RNA is to directly gouge tissue from the blocks instead of sectioning the blocks (as recommended in the PAXgene Tissue RNA Kit protocol). Hopefully this approach will eliminate excess paraffin, while increasing the amount of input tissue. Once I have RNA from the entire suite of samples, I’ll check the RNA integrity via Bioanalyzer and then we’ll decide on a facility to use for high-throughput sequencing.

 

BS-Seq Illumina Data Assembly/Mapping

Currently, there are two projects that we have performed BS-Seq with (Crassostrea gigas larvae OA (2011) bisulfite sequencing and LSU C.virginica Oil Spill MBD BS Sequencing) and we’re struggling to align sequences to the C.gigas genome. Granted, the LSU samples are C.virginica, but the C.gigas larvae libraries are not aligning to the C.gigas genome via standard BLASTn or using a dedicated bisulfite mapper (e.g. BS-Map). I’m currently BLASTing a de-novo assembly of the C.gigas larvae OA 400ppm sequencing that Steven made against the NCBI nt DB in an attempt to assess the taxonomic distribution of the sequences we received back. I’ll also try using a different bisulfite mapper, bismark, that Mackenzie Gavery has previously used and has had better results with than BS-Map.

 

C.gigas Heat Stress MeDIP/BS-Seq

As part of Claire’s project, there’s still some BS-Seq data that would be nice to have to complement the data she generated via microarray. It would be nice to make a decision about how to proceed with the samples. However, part of our decision on how to proceed is governed by the results we get from the two projects above. Why do those two projects impact the decision(s) regarding this project? They impact this project because in the two projects above, we produced our own BS-Seq libraries. This is extremely cost effective. However, if we can’t obtain usable data from doing the library preps in-house, then that means we have to use an external service provider. Using an external company to do this is significantly more expensive. Additionally, not all companies can perform bisulfite treatment, which limits our choices (and, in turn, pricing options) on where to go for sequencing.

 

Miscellany

When I have some down time, I’ll continue working on migrating my Wikispaces notebook to this notebook. I only have one year left to go and it’d be great is all my notebook entries were here so they’d all be tagged/categorized and, thus, be more searchable. I’d also like to work on adding README files to our plethora of electronic data folders. Having these in place will greatly facilitate the ability of people to quickly and more easily figure out what these folders contain, file formats within those folders, etc. I also have a few computing tips/tricks that I’d like to add to our Github “Code” page. Oh, although this isn’t really lab related, I was asked to teach the Unix shell lesson (or, at least, part of it) at the next Software Carpentry Workshop that Ben Marwick is setting up at UW in early June. So, I’m thinking that I’ll try to incorporate some of the data handling stuff I’ve been tackling in lab in to the lesson I end up teaching. Additionally, going through the Software Carpentry materials will help reinforce some of the “fundamental” tasks that I can do with the shell (like find, cut and grep).

In the lab, I plan on sealing up our nearly overflowing “Broken Glass” box and establishing a new one. I need to autoclave, and dispose of, a couple of very full biohazard bags. I’m also going to vow that I will get Jonathan to finally obtain a successful PCR from his sea pen RNA.

RNA Isolation – Geoduck Gonad in Paraffin Histology Blocks

UPDATE 20150528: The RNA isolated in this notebook entry may have been consolidated on 20150528.

Last week’s RNA isolation failed for more than half of the samples I processed. I will re-isolate RNA from the following samples:

  • 02
  • 03
  • 04
  • 07
  • 08
  • 09
  • 35
  • 38
  • 46
  • 65
  • 67
  • 68

IMPORTANT:

Five 5μm sections were taken from each block. A new blade was used for each block.

Samples were then processed with the PAXgene Tissue RNA Kit in two groups of six.

Isolated RNA according to the PAXgene Tissue RNA Kit protocol with the following alterations:

  • “Max speed” spins were performed at 19,000g.
  • Tissue disruption was performed with the Disruptor Genie @ 45C for 15mins.
  • Shaking incubation step was performed with Disruptor Genie
  • Samples were eluted with 40μL of Buffer TR4, incubated @ 65C for 5mins, immediately placed on ice and quantified on the Roberts Lab NanoDrop1000.

Results:

 

 

 

Well, these results are very consistent with the data from the last isolation performed on these samples. This fact suggests that the problem lies with the tissue samples and not the isolation (since the isolation has been performed two separate times on these same samples and the results have come out virtually identical both times).

All samples with concentrations < 5ng/μL were discarded. The remaining samples were stored @ -80C in Shellfish RNA Box #5:

  • 35
  • 38
  • 65
  • 67

Will discuss with Steven, look at Grace’s notebook to review the preservation process for these samples, and review the PAXgene Tissue RNA Kit to see if it will accommodate a greater number of microtome sections to use for isolation.

 

 

RNA Isolation – Geoduck Gonad in Paraffin Histology Blocks

UPDATE 20150528: The RNA isolated in this notebook entry may have been consolidated on 20150528.

Isolated RNA from geoduck gonad previously preserved with the PAXgene Tissue Fixative and Stabilizer and then embedded in paraffin blocks. See Grace’s notebook for full details on samples and preservation.

RNA was isolated from the following samples using the PAXgene Tissue RNA Kit (Qiagen) from the following geoduck sample blocks:

  • 02
  • 03
  • 04
  • 07
  • 08
  • 09
  • 35
  • 38
  • 41
  • 46
  • 51
  • 65
  • 67
  • 68
  • 69
  • 70

IMPORTANT:

Five 5μm sections were taken from each block. A new blade was used for each block.

Samples were then processed with the PAXgene Tissue RNA Kit in two groups of eight.

Isolated RNA according to the PAXgene Tissue RNA Kit protocol with the following alterations:

  • “Max speed” spins were performed at 19,000g.
  • Tissue disruption was performed with the Disruptor Genie @ 45C for 15mins.
  • Shaking incubation step was performed with Disruptor Genie
  • Samples were eluted with 40μL of Buffer TR4, incubated @ 65C for 5mins, immediately placed on ice and quantified on the Roberts Lab NanoDrop1000.

Results:

 

 

Well, these results are certainly not good.

The first set of eight samples I processed yielded no RNA (except #38, which is only marginally better than nothing). All the samples (excluding #38) have been discarded.

The second set of eight samples I processed range from amazing to poor (#68 was barely worth keeping).

I’ll review the protocol, but at the moment I’m at a loss to explain why the first set of eight samples came up empty. Will perform another on these blocks on Monday. Grrrrr.

Samples were stored at -80C in Shellfish RNA Box #5.