After confirming that the DNA available for this project looked good, I performed bisulfite treatment on the following gDNA samples:

• 1NF11

• 1NF15

• 1NF16

• 1NF17

• 2NF5

• 2NF6

• 2NF7

• 2NF8

• NF2_6

• NF2_18

• M2

• M3

Sample names breakdown like this:

1NF#

1 = Fidalgo Bay outplants

NF = Fidalgo Bay broodstock origination

= Sample number

2NF#

Same as above, but:

2 = Oyster Bay outplants

NF2_# (Oysters grown in Oyster Bay; DNA provided by Katherine Silliman)

NF2 = Fidalgo Bay broodstock origination, family #2

= Sample number

M2/M3 = C.gigas from Katie Lotterhos

Followed the guidelines of the [TruSeq DNA Methylation Library Prep Guide (Illumina)(https://github.com/sr320/LabDocs/blob/master/protocols/Commercial_Protocols/Illumina_truseq-dna-methylation-library-prep-guide-15066014-a.pdf).

Used the [EZ DNA Methylation-Gold Kit (ZymoResearch)(https://github.com/sr320/LabDocs/blob/master/protocols/Commercial_Protocols/ZymoResearch_EZ_DNA_Methylation-Gold_Kit_d5005i.pdf) according to the manufacturer’s protocol with the following changes/notes:

• Used 100ng DNA (per Illumina recs; Zymo recommends at least 200ng for “optimal results”).

• Thermal cycling was performed in 0.5mL thin-wall tubes in a PTC-200 (MJ Research) using a heated lid

• Centrifugations were performed at 13,000g

• Desulphonation incubation for 20mins.

DNA quantity calculations are here (Google Sheet): 20151218_oly_bisulfite_calcs

Samples were stored @ -20C. Will check samples via Bioanalyzer before proceeding to library construction.