Tag Archives: Oyster Bay

Epigenetic variation of two populations grown at common site

In a different experiment compared to when Fidalgo siblings were outplanted at two sites, we also examined Hood Canal (HC) and Oyster Bay (SS/South Sound) grown at Clam Bay (Manchester). Descriptor.

These were the oysters Katherine Silliman spawned in the summer of 2015 and represent seed Jake outplanted years ago.

This was run against the BGI scaffolds >10k.
BSMAP-06-BGIv002_1CC19CB1.png

The results are quite interesting.
RStudio_1CC19CEC.png

The full notebook can be found at https://github.com/sr320/nb-2016/blob/master/O_lurida/BSMAP-06-BGIv001.ipynb.

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DNA Isolation – Oly gDNA for BS-seq

Need DNA to prep our own libraries for bisulfite-treated high-throughput sequencing (BS-seq).

Isolated gDNA from the following tissue samples stored in RNAlater (tissue was not weighed) using DNAzol:

2NF1
2NF2
2NF3
2NF4
2NF5
2NF6
2NF7
2NF8
1NF11
1NF12
1NF13
1NF14
1NF15
1NF16
1NF17
1NF18

The sample coding breaks down as follows (see the project wiki for a full explanation):

2NF#

2 = Oysters outplanted in Fidalgo Bay

NF = Broodstock originated in Fidalgo Bay

= Sample number

1NF#

1 = Oysters outplanted in Oyster Bay

NF = Broodstock originated in Fidalgo Bay

= Sample number

 

DNA was isolated in the following manner:

  • Homogenized tissues in 500μL of DNAzol (Molecular Research Center; MRC).
  • Added additional 500μL of DNAzol.
  • Added 10μL of RNase A (10mg/mL, ThermoFisher); incubated 10mins @ RT.
  • Added 300μL of chloroform and mixed moderately fast by hand.
  • Incubated 5mins @ RT.
  • Centrifuged 12,000g, 10mins, RT.
  • Transferred aqueous phase to clean tube.
  • Added 500μL of 100% EtOH and mixed by inversion.
  • Pelleted DNA 5,000g, 5mins @ RT.
  • Performed 3 washes w/70% EtOH.
  • Dried pellet 3mins.
  • Resuspended in 100μL of Buffer EB (Qiagen).
  • Centrifuged 12,000g, 10mins, RT to pellet insoluble material.
  • Transferred supe to clean tube.

The samples were quantified using the Qubit dsDNA BR reagents (Invitrogen) according to the manufacturer’s protocol and used 1μL of sample for measurement.

Results:

Qubit data (Google Sheet): 20151216_Oly_gDNA_qubit_quants

SAMPLE CONCENTRATION (ng/μL)
2NF1 76.4
2NF2 175
2NF3 690
2NF4 11.7
2NF5 142
2NF6 244
2NF7 25
2NF8 456
1NF11 182
1NF12 432
1NF13 155
1NF14 21
1NF15 244
1NF16 112
1NF17 25.2
1NF18 278

 

Will run samples on gel tomorrow to evaluate gDNA integrity.

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MBD Enrichment – Sonicated Olympia Oyster gDNA

Olympia oyster gDNA that had previously been sonicated and fragmented was enriched for the methylated fragments using the MethylMiner Methylated DNA Enrichment Kit (Invitrogen).

Prepared the following components:

  • 20mL 1x Bind/Wash Buffer (4mL 5x Bind/Wash Buffer + 16mL H2O)
  • 640μL of beads (35μL of beads x 18 samples )
  • 200μL MBD-Biotin Protein (63μL MBD-Biotin Protein + 137μL 1x Bind/Wash Buffer)

Followed the manufacturer’s protocol for input DNA quantities 1μg – 10μg.

Used single fraction, high salt elution.

Neglected to account for the control reaction during initial set up and did not have sufficient quantities of beads to run a control reaction.

The table below provides the individual sample volumes and the volumes of the buffer, beads, H2O for the MBD capture reactions.

Samples listed with “NA” were not processed because they did not fragment during sonication.

Sample Volume (μL) Buffer/Beads (μL) H2O (μL) Total (μL)
hc1_2B 75 135 290 500
hc1_4B 90 135 275 500
hc2_15B 75 135 290 500
hc2_17 75 135 290 500
hc3_1 75 135 290 500
hc3_5 75 135 290 500
hc3_7 70 135 295 500
hc3_9 NA NA NA NA
hc3_10 70 135 295 500
hc3_11 70 135 295 500
ss2_9B 190 135 175 500
ss2_14B 195 135 170 500
ss2_18B 195 135 170 500
ss3_3B 190 135 175 500
ss3_4B NA NA NA NA
ss3_14B 195 135 170 500
ss3_15B 195 135 170 500
ss3_16B 195 135 170 500
ss3_20 135 135 230 500
ss5_18 75 135 290 500

 

Non-captured & wash fractions were pooled into single samples and stored @ -20C.

MBD fraction was EtOH precipitated according to the manufacturer’s protocol and incubate O/N @ -80C.

 

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DNA Sonication – Oly gDNA for MBD

In preparation for MBD enrichment, fragmented Olympia oyster gDNA with a target size of ~350bp. Used the Seeb Lab’s Bioruptor 300 (Diagenode) sonicator.

After sonication, samples were run on a the Seeb Lab’s 2100 Bioanalyzer (Agilent) on DNA 12000 chips.

Results:

HOOD CANAL SAMPLES

 

OYSTER BAY SAMPLES

More detailed analysis (including average fragment size for each samples) will be coming soon…

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DNA Sonication – Oly gDNA for MBD

In preparation for MBD enrichment, fragmented Olympia oyster gDNA with a target size of ~350bp.

Genomic DNA samples were isolated and provided to us by Katherine Silliman at UIC. Selected samples will compare Hood Canal (HC) and Oyster Bay (SS, South Sound) populations.

Used the Seeb Lab’s Bioruptor 300 (Diagenode) sonicator.

After sonication, samples were run on a the Seeb Lab’s 2100 Bioanalyzer (Agilent) on DNA 12000 chips.

Results:

HOOD CANAL SAMPLES

 

OYSTER BAY SAMPLES

More detailed analysis (including average fragment size for each samples) will be coming soon…

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