# 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

# 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

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.

#!/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&amp;cntrl=1160641846&amp;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&amp;cntrl=505010539&amp;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&amp;cntrl=636513375&amp;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&amp;cntrl=1472734408&amp;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&amp;cntrl=948605937&amp;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&amp;cntrl=1810346594&amp;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&amp;cntrl=424477466&amp;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&amp;cntrl=630586816&amp;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&amp;cntrl=1392201335&amp;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&amp;cntrl=1598310685&amp;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&amp;cntrl=868072864&amp;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&amp;cntrl=1074182214&amp;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 – 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

# 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 spec-ing all the samples because I noticed that the elution tubes had pellets in them! When I mix the tube prior to spec-ing (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…

# Illumina RNAseq Library Construction – 32 C.gigas Individuals

Took heat-fragmented RNA provided by Emma (see Emma’s Notebook, 7/3/2011) and proceeded to make first strand cDNA, as described in the Eli Meyer protocol for Illumina HiSeq. Master mix calcs are here. Samples were stored @ -20C after the reverse transcription and library construction will be continued tomorrow.

# Oligo Reconstitution – Illumina RNAseq Library Oligos and Barcodes

Reconstituted all of the oligos and barcodes for library construction in TE (pH = 8.0) to a final concentration of 100uM. Created 10uM working stocks of all oligos and barcodes. All samples (stocks and working stocks) are stored @ -80C in their own box (Illumina Library Oligos & Barcodes) due to the fact that one of the oligos is an RNA oligo and requires storage at -80C.

# Chloroform Clean Up – Lexie’s QPX RNA from 20110504

After submission of QPX samples to HTGU for Illumina library prep yesterday, I was notified that there was insufficient RNA for the QPX RNA samples. I checked the source RNA on the Roberts Lab NanoDrop1000 and determined that they had high phenol contamination (large peak at 270nm), which results in a large exaggeration in the OD260 absorbance (NanoDrop1000 report[JPEG]; notice terrible OD260/280 ratios; did not save screen shot of absorbance peaks.). As such, the concentrations that Lexie had listed in her notebook for these samples are highly inaccurate and highly inflated. To remove the phenol, I brought all of her QPX RNA samples from 20110504 up to ~200uL with 0.1%DEPC-H2O, added 200uL of chloroform, vortexed for 30s, spun at 12,500g RT for 15mins, and transferred aqueous phase to new tube. Then performed an ethanol precipitation on the aqueous phase. Added 0.1 vols of 3.0M sodium acetate (pH = 5.2), 2.5 vols of 100% EtOH, mixed and incubated at -20C for 1hr. Pelleted RNA by spinning at 16,000g 4C for 15mins.

Results:

As suspected, most of these samples have absolutely no RNA in them. However, the samples that do (the “Control” samples), look great! Pooled 2ug each of the RT Control a & b samples and pooled 2ug each of the 10C Control a & b samples (which are ATCC). Calculations are here. Will take them down to HTGU tomorrow to replace the bad samples that were provided yesterday.