Finished the ethanol precipitation as described in the MethylMiner (Invitrogen) manual which Yaamini had previously initiated: https://yaaminiv.github.io/Virginica-MBDSeq-Day4/

Samples were resuspended in 25μL of Buffer EB (Qiagen) and transferred to 0.5mL snap cap tubes. All tubes were labeled as: MBD CV #

Quantified the Crassostrea virginica MBD-enriched DNA with the Qubit 3.0 (ThermoFisher) and the Qubit dsDNA High Sensitivity (HS) Kit (ThermoFisher).

Used 1uL of template DNA.

Results:

Quantification Spreadsheet (Google Sheet):20180207_qubit_DNA_HS_MBD_virginica

One sample (MBD CV 106) may not be usable due to low yield. However, the remainder should work fine.

I’ve sent them all to ZymoResearch for bisulfite treatment, library construction, and Illumina sequencing.

FedEx tracking: 771429590026

Sent Crassostrea virginica samples to Qiagen, as part of the collaboration we have with them for testing their new bisulfite conversion kit on various reduced representation DNA.

Samples were sent on dry ice via FedEx International Priority: 771231112481

Here are the samples I sent:

gDNA C.virginica – Genomic DNA, 20uL, 58.4ng/uL
MBD 1 virginica – Fragmented (~400bp average size), MBD-enriched, 25uL, 18.3ng/uL
MBD 2 virginica – Fragmented (~400bp average size), MBD-enriched, 25uL, 19.6ng/uL
MspI 1 virginica – gDNA digested with MspI, 25uL, 53.4ng/uL
MspI 2 virginica – gDNA digested with MspI, 25uL, 31.0ng/uL

Genomic DNA was isolated from mantle tissue using the E.Z.N.A. Mollusc DNA Kit (Omega). DNA was eluted with the Elution Buffer supplied with the kit.

MBD enrichment was performed using the MethylMiner Methylated DNA Enrichment Kit (Invitrogen). DNA was resuspended in Buffer EB (Qiagen).

MspI digestions were performed using MspI (NEB) and were subjected to a phenol:chloroform cleanup, post-digestion. DNA was resuspended in Buffer EB (Qiagen).

No tests have been performed on the samples to evaluate the presence/absence of non-oyster DNA.

Reference genome is available here: https://www.ncbi.nlm.nih.gov/assembly/GCF_002022765.2/

Notebook entries:

gDNA Isolation:

http://onsnetwork.org/kubu4/2017/12/11/dna-isolation-quantification-crassotrea-virginica-mantle-gdna/

MBD:

DNA Shearing and Bioanalyzer assessments: http://onsnetwork.org/kubu4/2017/12/11/dna-sonication-bioanalzyer-c-virginica-gdna-for-medip/
Day 1: http://onsnetwork.org/kubu4/2018/01/08/mbd-enrichment-crassostrea-virginica-sheared-dna-day-1/
Day 2: http://onsnetwork.org/kubu4/2018/01/09/mbd-enrichment-crassostrea-virginica-sheared-dna-day-2/
Day 3: http://onsnetwork.org/kubu4/2018/01/10/mbd-enrichment-crassostrea-virginica-sheared-dna-day-3/
Quants: http://onsnetwork.org/kubu4/2018/01/10/dna-quantification-c-virginica-mbd-enriched-dna/

MspI Digestion:

Digestion: http://onsnetwork.org/kubu4/2018/01/11/restriction-digestion-mspi-on-crassotrea-virginica-gdna/
Phenol:Chloroform cleanup: http://onsnetwork.org/kubu4/2018/01/11/phenolchloroform-extractions-and-etoh-precipitations-mspi-digestions-of-c-virginica-dna-from-earlier-today/
Quants: http://onsnetwork.org/kubu4/2018/01/11/dna-quantification-mspi-digested-crassostrea-virginica-gdna/

Continuing Illumina’s generous efforts to use our geoduck samples to test out the robustness of their emerging sequencing technologies, they have requested we send them some more geoduck tissue so that they can try to isolate higher molecular weight DNA to complete the genome sequencing efforts using the 10x genomics sequencing platform.

The previous set of tissues sent did not yield DNA with high enough molecular weight.

This time, I pulverized ctenidia and foot tissues under liquid nitrogen with mortar and pestle, in hopes of improving the efficiency of the extraction process they’re using.

I sent pulverized tissue on dry ice. Tissue was collected by Brent & Steven on 20150811.

FedEx tracking: 770975522511

Continuing Illumina’s generous efforts to use our geoduck samples to test out the robustness of their emerging sequencing technologies, they have requested we send them some more geoduck tissue so that they can try to isolate higher molecular weight DNA to complete the genome sequencing efforts using the 10x genomics sequencing platform.

I sent three frozen pieces of geoduck foot, ctenidia, & adductor muscle tissue on dry ice. Tissue was collected by Brent & Steven on 20150811.

FedEx tracking: 770796037903

Continuing Illumina’s generous efforts to use our geoduck samples to test out the robustness of their emerging sequencing technologies, they have requested we send them some geoduck tissue so that they can try to complete the genome sequencing efforts using the 10x genomics sequencing platform.

I sent two frozen pieces (~28mg each) of geoduck ctendia tissue on dry ice. Tissue was collected by Brent & Steven on 20150811.

FedEx tracking: 770129114978

Sent the following RNA to Katherine Silliman at the Univ. of Chicago for RNAseq. All RNA was isolated on 20170719, except SN-10-16, which was isolated on 20170710.

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

All samples were sent on dry ice via FedEx Standard Overnight: 779698651635

Sent 10μg of the geoduck gDNA I isolated earlier today to Illumina on dry ice via FedEx Standard Overnight service.

Sent the following samples to Illumina for possible selection in a new pilot sequencing platform they’re working on.

The 12 samples will be used for RNAseq for genome annotation – numbers indicate desired sequencing priority.

Juvenile and larval samples were from Hollie Putnam (see links below for more info).

Other tissue was from a single, adult geoduck, collected by Brent & Steven on 20150811.

1. Gonad
2. Heart
3. Ctenidia
4. Juvenile OA exposure (super low)  (EPI_115, EPI_116)
5. Juvenile ambient exposure (ambient treatment) (EPI_123, EPI_124)
6. Larvae day 0 (EPI_74, EPI_75)
7. Larvae day 5 (EPI_99)
8. Crystalline style
9. Byssus gland
10. Mantle
11. Labial palps
12. Juvenile OA exposure – low treatment (EPI_107, EPI_108)

In addition to the above 12 samples, ~1.5μg of geoduck gDNA (isolated this morning) was sent.

Hollie Putnam asked me to help her get samples ready for submission for Illumina sequencing at Genewiz.

She had previously prepared reduced representation bisulfite libraries on 20161215.

She also prepped a whole genome library on 20161201 – specifically sample EPI_135 WG.

She needed these samples combined in to four separate pools. However, Pool 5 was to be pooled with a total of five samples, including EPI_135 WG. She asked that the EPI_135 WG sample make up 50% of the DNA in the pool.

Using her previously determined sample concentrations, I pooled the libraries in equal quantities.

Calculations (Google Sheet): 20161219_hollie_library_pool_calcs

The pool volumes are high and, the calculated pool concentrations are low. Due to time limitations on our end, it was not feasible for me to SpeedVac these down to achieve the target concentration of 10nM. I’ve notified Hollie and asked her to see if Genewiz will perform that service.

Samples were shipped on dry ice to Genewiz via FedEx Standard Overnight.

Submitted 10μg (30.7μL) of the O.lurida gDNA I isolated on 20161214 to the UW PacBio facility – Order #450.

Sequencing will be 10 SMRT cells. Turnaround time is ~7-8 weeks for UW customers (UW customers get queue priority).