LCM Zymo Trio WGBS Library Prep

Protocol link here

Samples: Testing 2 of the 10 extracted on 9/13

Zoe metadata link

Tapestation concentrations:

sample_id	concentration	DIN
#9 (Frag B)	4.37 ng/uL	3.1
#15 (Frag C)	3.80 ng/uL	2.4

Library Prep

• Rest of DNA (~9.5 uL for each sample) brought up to 20 uL with Tris buffer, with non-methylated DNA (E. coli DNA) spike-in
  • 100 pg of non-fragmented E. coli DNA was added to each sample, 0.4 uL of a 0.25 ng/uL dilution of the provided E. coli DNA
• Processed using EZ Methylation Gold Kit for Bisulfite Conversion (standard thermocycler settings), cleaned according to protocol and eluted in 19 uL DNA elution buffer
• Did 20 PCR cycles during Index PCR
• Adjusted protocol from Zymo Rep:
  • Following index PCR, please follow the below steps for bead cleanup before the written Appendix A protocol:
    • Add 60 µL of Select-a-Size MagBeads and mix thoroughly. Incubate for 5 minutes at room temperature.
    • Pellet on a magnetic stand for 3 minutes or until the supernatant is clear. Carefully remove and discard the supernatant.
    • Remove the sample from the magnetic stand and add 50 µL of DNA Elution Buffer, mixing thoroughly by pipetting.
    • Add 60 µL Library Binding Solution to the resuspended sample+bead and mix thoroughly. Incubate for 5 minutes at room temperature.
    • Proceed to the Appendix A cleanup protocol beginning with Step 2 (placing tube on magnetic stand), and carry out the remaining steps as usual.

QC

Run to visualize libraries. Here’s an example of what the library should look like on a Tapestation:

“If adapter dimers are present, they will form an approximately 130-180 bp band. Yields will vary depending on the total quantity and quality of sample input DNA.

Results

This is really interesting. There seems to be better concentration, but more adapter dimer. Will write Zymo.

Compare to sequenced libraries (pre extra cleanup):

Full results can be found here