Tag Archives: library prep

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

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Library Prep – Oly Oyster gDNA-01 RAD

Used gel-purified, size-selected DNA from yesterday to prepare the RAD library using the Kappa LTP Kit:

http://eagle.fish.washington.edu/trilobite/Sites_genefish_100112/Steven/Commercial%20Protocols/KAPA_Biosystems%20-%20KAPA_LTP_Library_Preparation_Kit_TDS.pdf

The protocol was followed with the following changes:

– Section 8

Skipped entirely

– Section 9.1

Used 10uL of library DNA (instead of 20uL)

Used 1uL of mixed primer set (instead of 5uL)

– Section 9.2

Performed 12 cycles of PCR protocol. This was Carita’s recommendation and experience with using the Kappa LTP Kit for RAD library construction.

Sample was eluted from the AMPure beads with 15uL of Buffer EB (Qiagen) and stored @ -20C.

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DNA Shearing & Size Selection – Oly Oyster gDNA RAD P1 Adapters (from 20141105)

Pooled “low quality” samples and pooled “high quality” samples separately (in 1.5mL snap cap tubes) prior to shearing to improve chances of getting similar DNA size ranges.

Samples were selected based on the gels run by Steven on Oct. 17, 2014: http://sr320.tumblr.com/

Low quality samples (5uL from each):

All rows, columns 1 -9

Higher quality samples (5uL from each):

All rows, columns 10 -12

Sheared each samples with the following cycling protocols on the Biorupter Plus (Diagenode):

Low

  • 3 cycles of:
  • 30 seconds on
  • 59 seconds off

 

High

4 cycles of:

  • 30 seconds on
  • 59 seconds off

 

Ran a subset of sheared gDNA (5uL from each pool) on gel to verify final size range:

Gel loading:

Lane 1 – O’GeneRuler 100bp Ladder (ThermoFisher)

Lane 2 – Low quality

Lane 3 – Higher quality

I neglected to run a set of un-sheared DNA.

Both samples appear to have an average size of 200 – 400bp.

After confirming satisfactory shearing, the two samples were combined and run on a 1% agarose low TAE gel (stained with EtBr) for size selection.

O’GeneRuler 100bp Ladder (ThermoFisher)

O’GeneRuler 100bp Ladder (ThermoFisher)

Size range of sheared DNA from 300 – 500bp was excised from gel.

 

Gel fragment weighed 254mg.

Purified using MiniElute Gel Extraction Kit (Qiagen).

Added three volumes (762uL) of Buffer QG to gel slice.

Incubated ~10mins on rotator until gel slice was fully dissolved.

Added one gel slice volume (254uL) of isopropanol; inverted multiple times to mix.

Added 700uL to MiniElute column; spun max speed (~16,000g) 1min; discarded flow-through.

Added remainder of sample to MiniElute column; spun max speed (~16,000g) 1min; discarded flow-through.

Added 500uL of Buffer QG to MiniElute column; spun max speed (~16,000g) 1min; discarded flow-through.

Added 750uL of Buffer PE to MiniElute column; incubated @ RT for 5mins; spun max speed (~16,000g) 1min; discarded flow-through.

Spun MinElute column spun max speed (~16,000g) 1min; transferred column to clean 2.0mL tube.

Added 50uL of Buffer EB to column, incubated @ RT for 5mins and spun max speed (~16,000g) 1min; discarded column.

Stored sample @ 4C.

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Ligation – Illumina P1 Adapters for Oly Oyster gDNA-01 RAD Sequencing (from 20141031)

Made 25nM working stock from the 100nM stock adapter plate provided by Carita. Added 2uL of each adapter to corresponding well of SbfI digested DNA (e.g. DNA plate well A1 got the P1 adapter from well A1 in the adapter plate).

Created master mix of the ligation components and added 3uL to each well of SbfI-digested Oly gDNA.

Master mix calcs are here: 20141105 – Oly Oyster gDNA-01 Adapter Ligation

Incubated @20C for 60mins. Deactivated ligase @65C for 30mins. Stored @ 4C.

Adapter plate layout and sequences can be found here: 96_SbfI_RAD_adapter_sequences

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Restriction Digest – Oly Oyster gDNA-01 for RAD Sequencing (from 20141029)

Samples required two days of drying for all samples to fully dry down.

Reconstituted all samples in 20uL of PCR H2O.

Performed restriction digests:

Incubated at 37C for 90mins and then inactivated enzyme @ 80C for 20mins. Incubation was done in thermal cycler using heated lid. Plate was stored @ -20C.

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Illumina RNAseq Library Construction – 32 C.gigas Individuals

Took heat-fragmented RNA provided by Emma (see Emma’s Notebook, 7/3/2011) and proceeded to make first strand cDNA, as described in the Eli Meyer protocol for Illumina HiSeq. Master mix calcs are here. Samples were stored @ -20C after the reverse transcription and library construction will be continued tomorrow.

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SOLiD ePCR/Templated Bead Prep – Lake Trout Lean library

ePCR was performed following ABI’s “full scale” protocol, using 1pM of SOLiD cDNA library.

Templated bead preparation was performed according to the “full scale” protocol.

Bead counts are calculated as follows:

Avg bead count x # hemacytometer squares x volume in hemacytometer (uL) x dilution factor = beads/uL x suspension volume (uL) = total beads

Initial Bead count: (1:200 dilution)

Lean: 126, 138, 122, 138 Avg. = 131

Lean: 131 x 25 x 10 x 200 = 6.55×10^6 beads/uL x 200uL = 1.31×10^9 beads

Templated Bead counts (1:10 dilution)

Lean: 165, 171, 186, 160 Avg. = 170.5

170.5 x 25 x 10 x 10 = 426250 beads/uL x 400uL = 1.705×10^8 beads

Percent Recovery Templated Beads

Lean: (1.705×10^8 beads)/(1.31×10^9 beads) x 100 = 13.02% recovery

Results: Yield is significantly higher than the previous preparation performed with this sample. The percent recovery falls into the desired range of 5-15%, so things look good there, too. Will contact Rhonda and get info regarding when this and the other 7 samples can go on a run.

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SOLiD Templated Bead Prep – Yellow perch CT, WB and lake trout Lean libraries (continued from yesterday)

Templated bead preparation was performed according to the “full scale” protocol.

Bead counts are calculated as follows:

Avg bead count x # hemacytometer squares x volume in hemacytometer (uL) x dilution factor = beads/uL x suspension volume (uL) = total beads

Initial Bead counts: (1:200 dilution)

CT: 132, 133, 127, 136 Avg. = 132

WB: 127, 128, 119, 126 Avg. = 125

Lean: 121, 114, 132, 109 Avg. = 119

CT: 132 x 25 x 10 x 200 = 6.6×10^6 beads/uL x 200uL = 1.32×10^9 beads

WB: 125 x 25 x 10 x 200 = 6.25×10^6 beads/uL x 200uL = 1.25×10^9 beads

Lean: 119 x 25 x 10 x 200 = 5.95×10^6 beads/uL x 200uL = 1.19×10^9 beads

Templated Bead counts (1:10 dilution)

CT: 91, 80, 100, 78 Avg. = 87.25

WB: 69, 70, 75, 65 Avg. = 69.75

Lean: 40, 52, 48, 46 Avg. = 46.5

CT: 87.25 x 25 x 10 x 10 = 218125 beads/uL x 400uL = 8.7525×10^7 beads

WB: 39.75 x 25 x 10 x 10 = 174375 beads/uL x 400uL = 6.975×10^7 beads

Lean: 46.5 x 25 x 10 x 10 = 116250 beads/uL x 400uL = 4.65×10^7 beads

Percent Recovery Templated Beads

CT: (8.7252×10^7 beads)/(1.32×10^9 beads) x 100 = 6.61%

WB: (6.975×10^7 beads)/(1.25×10^9 beads) x 100 = 5.58%

Lean: (4.65×10^7 beads)/(1.19×10^9 beads) x 100 = 3.91%

Results: Everything looks pretty darn good. One mild concern, however, is the yield from the the Lean library. An 8-well slide requires 41 million beads for a run. Additionally, I believe 15 million are needed for a WFA (quality check, pre-run). This means that the Lean prep is nearly 10 million beads short of what is necessary for a “complete” run of this sample. Will send the numbers to Rhonda and see what her opinion is and what she suggests to do. But, based on the percent recovery, all the samples should be really high quality (extremely few polyclonal beads).

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SOLiD ePCRs – Yellow perch CT, WB and lake trout Lean libraries

Performed ePCRs on these samples from DATE, following the “full scale” protocol. A work flow analysis (WFA) run on these samples from the initial ePCRs/templated bead prep (DATE) revealed too many polyclonal beads, thus requiring them to be redone. ePCRs will be performed using 1.0pM (120pg/uL) of the SOLiD cDNA fragment libraries, instead of the 1.5pM (180pg/uL) used previously.

CT –

WB –

Lean -

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