Author Archives: kubu4

RNA Isolation – Olympia oyster gonad tissue in paraffin histology blocks

My previous go at this was a little premature – I didn’t wait for Laura to fully annotate her slides/blocks. Little did I know, the tissue was mostly visceral mass and, as such, I didn’t hit much in the way of actual gonad tissue. So, I’m redoing this, now that Grace has gone through and annotated the blocks to point out gonad tissue. SN-10-16 was sent to Katherine Silliman on 20170720.

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

 

RNA was isolated from the following samples using the PAXgene Tissue RNA Kit (Qiagen). Gouged samples from the blocks weighing ~10mg from each of the tissues and processed according the protocol for isolating RNA from blocks of paraffin-embedded tissues.

Background on all of this is in this GitHub Issue

NF-10-22
NF-10-23
NF-10-24
NF-10-26
NF-10-28
NF-10-30
SN-10-16
SN-10-17
SN-10-20
SN-10-25
SN-10-26
SN-10-31

IMPORTANT:

  • Prior to beginning, I prepared an aliquot of Buffer TR1 by adding 40μL of β-mercaptoethanol (β-ME) to 4000μL of Buffer TR1)

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

  • “Max speed” spins were performed at 20,000g.
  • Tissue disruption was performed by adding ~25-50 glass beads (425 – 600μm diameter) with the Disruptor Genie @ 45C for 15mins (in the Friedman Lab).
  • Samples were eluted with 27μL of Buffer TR4 x 2, incubated @ 65C for 5mins, immediately placed on ice.

 

Results:

Samples were not quantified due to lack of proper RNA Qubit assay AND the computer that our NanoDrop1000 is hooked up to is dead. Will have Katherine Silliman perform quantification.

Samples were stored at -80C temporarily.

Samples will be sent to Katherine Silliman for high-throughput library construction and sequencing once I hear back from her regarding her availability to receive the samples.

Share

PCR – pCR2.1/OsHV-1_ORF117 Colony Screens

Screened five colonies from yesterday’s transformation via PCR using M13 primers.

I don’t have any sequence for the actual insert, so am relying on assessing empty vector vs vector with insert, based on PCR amplicon size.

Master mix calcs:

2x GoTaq Green Master Mix: 80uL
M13 forward: 4uL
M13 reverse: 4uL
H2O: 88uL

Added 20uL to each PCR tube.

Colonies were selected randomly, streaked on a new LB Amp100 plate with a sterile pipet tip, and then added to the PCR tube.

Cycling params:

1 cycle

95C – 10mins

30 cycles:

95C – 15s
55C – 15s
72C – 30s

1 cycle

72C – 5mins

PCR reactions were run on a 1% agarose 1xTBE gel + EtBr.

5uL of O’GeneRuler DNA Ladder Mix was loaded for sizing.

Results:


 

 

 

 

 

 

 

Well, these results are confusing. Immediate conclusion is that all colonies screened are empty, due to the small size of the amplicons produced (<100bp). However, looking at a vector map of pCR2.1 (the vector that the OsHV-1 ORF117 is supposedly cloned in), there are ~200bp between the M13 forward and M13 reverse primers. So, even an empty vector should produce an amplicon larger than what is seen on this gel.

I’ll contact Tim Green to see if he can provide any insight (and/or any actual sequence for OsHV-1 ORF117 so that I can order an insert specific primer to aid in confirmation).

Share

Transformation – pCR2.1/OsHV-1_ORF117 into One Shot Top10 Chemically Competent Cells

Used 5uL of the pCR2.1/OsHV-1_ORF117 plasmid provided by Tim Green to transform a single aliquot of One Shot Top10 Chemically Competent Cells (Invitrogen), according to the “Rapid Transformation” protocol.

Cells were plated on pre-warmed (37C) LB Amp100 plates with X-gal.

Plates were incubated overnight at 37C.

Results:

Looks good – ample colonies and no blue colonies (blue colonies = empty vector). Will screen a subset of the colonies via cPCR.

 

 

Share

RNA Isolation – Olympia oyster gonad tissue in paraffin histology blocks

UPDATE 20170712: The RNA I isolated below is from incorrect regions of tissue. I misunderstood exactly what this tissue was, and admittedly, jumped the gun. The tissue is actually collected from the visceral mass – which contains gonad (a small amount) and digestive gland (a large amount). The RNA isolated below will be stored in one of the Shellfish RNA boxes and I will isolate RNA from the correct regions indicated by Grace

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

 

RNA was isolated from the following samples using the PAXgene Tissue RNA Kit (Qiagen). Gouged samples from the blocks weighing ~10mg from each of the tissues and processed according the protocol for isolating RNA from blocks of paraffin-embedded tissues.

Tissue identification is available in this GitHub Issue

NF-10-22
NF-10-23
NF-10-24
NF-10-26
NF-10-28
NF-10-30
SN-10-16
SN-10-17
SN-10-20
SN-10-25
SN-10-26
SN-10-31

IMPORTANT:

  • Prior to beginning, I prepared an aliquot of Buffer TR1 by adding 40μL of β-mercaptoethanol (β-ME) to 4000μL of Buffer TR1).
  • Reconstituted DNase I with 550μL of RNase-free H2O. Aliquoted in 100μL volumes and stored @ -20C in the “-20C Kit Components” box.

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

  • “Max speed” spins were performed at 20,000g.
  • Tissue disruption was performed by adding ~25-50 glass beads (425 – 600μm diameter) with the Disruptor Genie @ 45C for 15mins (in the Friedman Lab).
  • Samples were eluted with 27μL of Buffer TR4 x 2, incubated @ 65C for 5mins, immediately placed on ice and quantified on the Roberts Lab Qubit 3.0 with the RNA High Sensitivity Assay (ThermoFisher Scientific) using 5μL of each sample.

Results:

Concentrations (Google Sheet): 20170710_RNA_qubit_oly_histo_blocks

Well, the good news is that there’s RNA from all the samples and it seems to be in relatively high concentrations!

The bad news is that the concentrations for 10 of the 12 samples were too high and outside the range of the Qubit RNA HS Assay! Since we don’t have the broad range RNA assay, I can’t properly quantify the remaining samples. However, these samples are being sent to Katherine Silliman at some point, so I’ll leave it up to her to quantify the samples. I’m also guessing that she’ll run them on a Bioanalyzer to assess their integrity prior to beginning library construction, so that will also yield concentrations for the samples.

Samples were stored at -80C temporarily.

Samples will be sent to Katherine Silliman for high-throughput library construction and sequencing once I hear back from her regarding her availability to receive the samples.

Share

Data Management – SRA Submission Olympia Oyster UW PacBio Data from 20170323

Submitted the FASTQ files from the UW PacBio Data from 20170323 to the NCBI sequence read archive (SRA).

FTP’d the data to NCBI’s servers, following their instructions. Briefly,

Change to the directory where the FASTQ files are (Owl/web/nightingales/O_lurida) and then initiate an FTP session:

ftp -i ftp-private.ncbi.nlm.nih.gov

Enter provided username/password, change to my designated uploads directory, create new folder dedicate to this particular upload. Then, upload all the files using the mput command:

mput *filtered_subreads*

SRA deets are below (assigned FASTQ files to existing BioProject and created a new BioSample). Will update post with SRA number when processing is complete on the NCBI end.

SRA: SRS2339870
Study: SRR5809355
BioProject: PRJNA316624
BioSample: SAMN07326085

Share

Data Management – Tarball of Olympia oyster UW PacBio Data from 20170323

I’d previously attempted to archive this data set on multiple occasions, across multiple days, but network dropouts kept killing my connection to the server (Owl) and, in turn, interrupting the tarball operation.

Today, I came in to a successful creation of the tarball of this PacBio data set (it only took 10hrs)! And, it’s a big file: 162GB!! Remember, that’s the compressed size!

Now, we’ll have to decide where we want to keep the tarball. I guess this’ll be part of our next data management plan discussions.

 

Share

Sample Annotation – Olympia oyster histology blocks (from Laura Spencer)

I’ve been asked to isolate RNA from some paraffin-embedded Olympia oyster gonad tissue.

Despite some excellent documentation by Laura Spencer (images of tissue layouts in histology cassettes and a corresponding cassette mapping key file), the histology facility seems to have flipped some things around and/or repositioned/split the contents of each cassette. This makes ID-ing the proper tissues tedious and, at times, difficult.

The list of tissues that needs to be processed is listed in this GitHub Issue #648. I’ve also added the list below:

NF-10 22
NF-10-23
NF-10-24
NF-10-26
NF-10-28
NF-10-30
SN-10-16
SN-10-17
SN-10-20
SN-10-25
SN-10-26
SN-10-31

Prior to beginning RNA isolations, I have annotated images of the histology blocks and will be waiting for Laura to confirm that my annotations are correct. I will be posting a link to this notebook entry in the GitHub issue listed above for her to view and wait for her confirmation.

UPDATE 201700707 – Laura has indicated that many of my annotations are incorrect. Katie has gone through and made proper identification: https://github.com/sr320/LabDocs/issues/648#issuecomment-313792588

 

Additionally, as indicated in the GitHub Issue above, histology block “Oly 14″ does not have a corresponding tissue cassette photo (containing sample NF-10 26). Without the original image, I don’t think I can make an accurate guess on how the tissues are oriented in the resulting two histo blocks (see below).

 

BLOCKS 5

 

BLOCK 6

 

 

BLOCK 9

 

 

BLOCK 10

 

 

BLOCKS 14 (unable to annotate at time of posting)

 

 

 

BLOCK 15

 

 

 

BLOCK 21

 

 

 

 

BLOCK 22

Share