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 – Claire’s C.gigas Female Gonad for Illumina Bisulfite Sequencing

Due to poor “tag counts” from the initial sequencing (DATE) and the re-sequencing (20131127) of this sample, the HTGU facility has concluded that the library is probably at fault. They will make a new library and do a quality control run on the new library. However, they have insufficient gDNA left to make a new library.

Isolated gDNA from Claire’s sample following the DNAzol protocol.

Transferred ~300uL of female C.gigas gonad from the source tube (ethanol-preserved) to a clean tube. Pelleted gonadal material by spinning 10,000g, 30seconds, @ RT. Decanted residual ethanol. Resuspended tissue in 500uL of DNAzol + 100ug of Proteinase K (Fermentas; 18.5mg/mL). Incubated on a rotator for ~6hrs. Proceeded according to DNAzol protocol. Resuspended final pellet in 100uL of Elution Buffer (Qiagen; EB). After resuspension, pelleted remaining debris 16,000g, 30seconds, @ RT. Transferred supernatant to clean tube and quantified on NanoDrop 1000.

CgF – 403.2ng/uL

Will bring tube to sequencing facility tomorrow morning.

EtOH Precipitation – LSU C.virginica Oil Spill MBD Continued (from 20141126)

Precipitation was continued according to the MethylMiner Methylated DNA Enrichment Kit (Invitrogen). Since I will need sample volumes of 24uL for the subsequent bisulfite conversion, I resuspended the samples in 29uL of water (will use 2.5uL x 2 reps for quantification).

Samples to be quantified:

NC = non-captured (i.e. non-methylated)

E = eluted (i.e. methylated)

  • HB2 NC
  • HB5 NC
  • HB16 NC
  • HB30 NC
  • NB3 NC
  • NB6 NC
  • NB11 NC
  • NB21 NC
  • HB2 E
  • HB5 E
  • HB16 E
  • HB30 E
  • NB3 E
  • NB6 E
  • NB11 E
  • NB21 E
  • Control NC
  • Control E

Samples were quantified using the Quant-IT BS Kit (Invitrogen) with a plate reader (BioTek). All samples were run in duplicate. Used 2.5uL of each sample for quantification.

Samples were stored in @ -20C (FTR 209) in the bisulfite seq box created by Claire for this project.

Results:

20141202_LSU_Virginica_MBD:

https://docs.google.com/spreadsheets/d/1NrrVmYsUQcstnrt4583mYN2PeVav54luyFvVUEkcjWE/edit?usp=sharing

Methylated DNA Enrichment (MBD) – LSU C.virginica Oil Spill gDNA

Enrichment was performed using the MethylMiner Methylated DNA Enrichment Kit (Invitrogen) according to the manufacturer’s protocol with the following changes:

– Used 25uL of Dynabeads M-280 (10uL/ug of input DNA) and 15uL of MBD-Biotin Protein (7uL/ug of input DNA).

– Followed the corresponding instructions for the volumes listed above and for quantities of input DNA > 1ug – 10ug

– A single elution with 2000mM NaCl was performed

– EtOH precipitation: Samples were incubated over the long weekend at -80C.

Gel – Sheared LSU C.virginica Oil Spill gDNA (from yesterday)

Ran ~250ng of sheared C.virginica gDNA from yesterday’s shearing.

Results:

Ladder used: O’GeneRuler 100bp Ladder (ThermoFisher)

The shearing is, surprisingly, very inconsistent across the samples. The target average fragment size was ~350bp. However, most of these samples are <250bp. The MethylMiner Kit (Invitrogen) suggests that an average fragment length of 100 – 200bp is ideal for short-read high-throughput sequencing, but we’re going to perform a bisulfite conversion on these which will result in some additional fragmentation, further reducing the average fragment size. Will proceed with methylated DNA enrichment.

DNA Shearing – LSU C.virginica Oil Spill gDNA

Used the remainder of the “sheared” samples (see today’s earlier entry; ~2750ng). Brought the volumes up to 80uL and transferred to 0.5mL snap cap tubes. The volume of 80uL was selected because it’s above the minimum volume required for shearing in 0.5mL tubes (10uL according to the Biorupter 300 manual) and the MethylMiner Kit (Invitrogen) requires the input DNA volume to be <= 80uL.

DNA was sheared with the following parameters:

Low power

30 cycles of:

30s on

30s off

Target average fragment size is ~350bp.

See tomorrow (20141126) for the gel.

Gel – Sheared gDNA

Ran ~250ng (out of 3000ng, according to Claire) of LSU C.virginica oil spill gDNA on a gel that was previously sheared by Claire to verify that shearing was successful.

Ran unsheared side-by-side with sheared gDNA for comparison.

Note: HB16 and NB3 did not have any unsheared gDNA left in their tubes, so nothing was run on a gel.

Results:

Ladder used: O’GeneRuler 100bp Ladder (ThermoFisher)

Well, it’s rather obvious that the initial shearing did NOT work. Will re-shear the samples.

UPDATE: Looking at the Biorupter (Diagenode) manual, it turns out that shearing samples in a 1.5mL tube (in which these were sheared) requires a minimum volume of 100uL. All the samples were far below this minimum volume. Additionally, the recommendations in the manual to reach the target size range are significantly longer (30 – 40 cycles) than what was applied (4 cycles). The combination of these two factors are likely the reason that shearing didn’t take place.

RNA Seq – C.gigas Total RNA from Claire’s Pre/Post Heat Shock

Shipped (dry ice) ~15ug of total RNA from each of the following samples to Genewiz, Inc for high throughput transcriptomic sequencing (Illumina HiSeq2500, 100bp, single end).

  • 2M – 33uL
  • 4M – 41uL
  • 6M – 15.6uL
  • 2M HS – 15uL
  • 4M HS – 12.2uL
  • 6M HS – 15.2uL

Here’s Claire’s notebook entry on their isolation and quantification:

http://claireeolson.blogspot.com/2014/10/rna-extractions.html