Category Archives: Crassostrea gigas larvae OA (2011) bisulfite sequencing

Bisulfite NGS Library Prep – LSU C.virginica Oil Spill MBD Bisulfite DNA and Emma’s C.gigas Larvae OA Bisulfite DNA (continued from yesterday)

Continued library prep from yesterday. Set up Library Amplification according to the protocol. The samples received the following Barcode Indices:

  • HB2 – 1 (ATCACG)
  • HB5 – 2 (CGATGT)
  • HB16 – 3 (TTAGGC)
  • HB30 – 4 (TGACCA)
  • NB3 – 5 (ACAGTG)
  • NB6 – 6 (GCCAAT)
  • NB11 – 7 (CAGATC)
  • NB21 – 12 (CTTGTA)
  • 1A1 – 2 (CGATGT)
  • 1A2 – 1 (ATCACG)
  • 6A1 – 4 (TGACCA)
  • 6A2 – 5 (ACAGTG)
  • 103B1 – 6 (GCCAAT)
  • 103B2 – 7 (CAGATC)
  • 105A4 – 12 (CTTGTA)
  • 105A5 – 11 (GGCTAC)

Due to differences in input DNA quantities, samples were run with different numbers of thermal cycles.

13 thermal cycles were run for the following samples:

  • 1A1
  • 105A4
  • 105A5

22 thermal cycles were run for the following samples:

  • HB2
  • HB5
  • HB16
  • HB30
  • NB3
  • NB6
  • NB11
  • NB21
  • 1A2
  • 6A1
  • 6A2
  • 103B1
  • 103B2

Samples were quantified with 1uL of each sample using the Quant-iT dsDNA BR Kit (Invitrogen). Used 5uL of each standard and standards were run in duplicate.

Results:

Bisulfite NGS Library Prep – LSU C.virginica Oil Spill Bisulfite DNA and Emma’s C.gigas Larvae OA Bisulfite DNA

Constructed next generation libraries (Illumina) using the bisulfite-treated DNA from yesterday using the EpiNext Post-Bisulfite DNA Library Preparation Kit – Illumina (Epigentek). Samples were processed according to the manufacturer’s protocol up to Section 8 (Library Amplification) with the following changes:

– Skipped Section 7.1 (recommended to do so in the protocol due to low quantity of input DNA)

Samples were stored O/N @ -20C.

dA Tailing Master Mix

10x Tailing Buffer 1.5uL x 17.6 = 26.4uL

Klenow 1uL x 17.6 = 17.6uL

H2O 0.5uL x 17.6 = 8.8uL

Add 3uL of master mix to each sample

Adaptor Ligation

2x Ligation Buffer 17uL x 17.6 – 299.2uL

T4 DNA Ligase 1uL x 17.6uL = 17.6uL

Adaptors 1uL x 17.6 = 17.6uL

Added 19uL of master mix to each sample

dsDNA Conversion Master Mix

5x Conversion Buffer 4uL x 17.6 = 70.4uL

C.P. 2uL x 17.6 = 35.2uL

H2O 3uL x 18.6 = 52.8uL

Add 9uL of master mix to each sample

End Repair

10x Buffer 2uL x 17.6 = 35.2uL

Enzyme 1uL x 17.6 = 17.6uL

H2O 5uL x 17.6 = 88uL

Added 8uL of master mix to each sample

Bisulfite Conversion – LSU C.virginica Oil Spill MBD DNA and Emma’s C.gigas Larvae OA DNA

Performed bisulfite conversion on MBD DNA samples from LSU C.virginica oil spill samples (see 201411202 and 20141126) and Emma’s C.gigas larvae OA DNA samples (see 20141121) with the Methylamp DNA Modification Kit (Epigentek).

Added 4uL of H2O to each of Emma’s DNA samples to bring them up to 24uL.

Samples were processed according to the manufacturer’s protocol.

Samples were eluted with 10uL of Solution R6 and stored @ -20C.

DNA Isolation – C.gigas Larvae from Emma OA Experiments

Isolated gDNA using DNazol from the following larval samples for potential MBD selection and bisulfite sequencing:

– Added 500uL of DNAzol to each tube, transferred to 1.5mL tube and homogenized with disposable pestles

– Added additional 500uL DNAzol to each homogenized sample and mixed by inversion.

– Incubated 10mins at RT

– Pelleted debris by spinning 10,000g, 10mins, @ RT

– Transferred supes to new tubes

– Added 500uL of 100% EtOH to each; mixed by inversion; incubated 10mins @ RT

– Pelleted DNA by spinning 5,000g, 4mins, @ RT

– Discarded supes

– Washed DNA with 1mL 70% DNAzol/30% EtOH solution

– Spun 1000g, 1min, @ RT

– Discard supes

– Washed DNA with 1mL 75% EtOH

– Spun 1000g, 1min, @ RT

– Discarded supes

– Spun 1000g, 1min, @ RT

– Removed residual EtOH with pipette; air dried samples for 5mins @ RT

– Added 20uL of Trish-HCl (pH = 8.0) to each sample; incubated 10mins @ RT; flicked tubes to help dissolve

– Spun 12,000g, 10mins, @ RT

– Transferred supes to new tubes

– Spec’d on NanoDrop 1000 (ThermoFisher) and recovered solution from each sample due to limited sample volume

Results: