Tag Archives: WB

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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ePCR SOLiD Libraries – Yellow perch PQ, WB and Lake Trout Lean libraries (from 20100408)

ePCR was performed for the above three mentioned SOLiD libraries using 1.5pM (180 pg/uL) of cDNA, according to the ABI “full scale” ePCR protocol. ePCRs were stored @ 4C until ready for the emulsion breaking step.

Amounts of cDNA used to make dilutions (in 1x Low TE Buffer) of 180pg/uL:

PQ (20.77ng/uL): 4.33uL in 500uL

WB (16.81ng/uL): 2.14uL in 200uL

Lean (53.49ng/uL): 1.68uL in 500uL

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cDNA clean up & Bioanalyzer for SOLiD Libraries – Abalone, Yellow Perch, Lake Trout, Herring

Amplified cDNA was cleaned up using the Invitrogen PureLink Micro Kit, but was done so according to Ambion’s Whole Transcriptome Analysis Kit protocol and then spec’d.

Results:

0.5uL was removed from each sample and mixed with 0.5uL to run on DNA 1000 chips on the Bioanalyzer 2100. The slideshow below shows the electropherograms from each sample. Each sample (to be considered worthy of moving to the next stage) should have <20% of the sample in the 25-150bp range. All 8 samples exhibit this and their peaks look very good. Will proceed to ePCR/templated bead prep next week.

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Gel Purification & PCR cDNA SOLiD Libraries – Abalone, Yellow Perch, Lake Trout, Herring

cDNA was gel purified according to Ambion’s Whole Transcriptome Analysis Kit. The appropriate regions (100 – 200bp) were excised and cut in to 4, 1x5mm pieces. The two “internal” pieces were transferred to individual PCR tubes. The “outer” pieces were transferred together to a 1.5mL snap cap tube and stored @ -20C.

Three images are below. The first two are the gels before excising the 100 – 200bp region of the gel. The third is the image of the SECOND gel after the specified region was excised. An image was not taken of Gel 1 after excision (whoops!).

Gel 1

 

Gel 2

NOTE: The WB sample in the gell above is actually a yellow perch sample, NOT an abalone sample!

 

Gel 2 AFTER EXCISION

NOTE: The WB sample in the gell above is actually a yellow perch sample, NOT an abalone sample!

 

In-gel PCR SOLiD Libraries – Abalone, Yellow Perch, Lake Trout, Herring

In-gel PCR was performed on the individual “internal” gel pieces that were excised, as described below from earlier today. PCR rxns/cycling were performed according to Ambion’s Whole Transcriptome Analysis Kit. PCR ran O/N. PCR master mix set up is here (bottom half of sheet).

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Reverse Transcription SOLiD Libraries – Abalone, Yellow Perch, Lake Trout, Herring

Samples were speedvac’d to dryness and resuspended in 3uL of nuclease-free H2O. Samples were then reversed transcribed according to Ambion’s Whole Transcriptome Analysis Kit. RT master mix set up is here (top portion of sheet).

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Hibridizaton/Ligation SOLiD Libraries – Abalone, Yellow Perch, Lake Trout, Herring

All 8 samples were hybridized/ligated according to Ambion’s Whole Transcriptome Analysis Kit using Adaptor A.

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SOLiD Library Prep – mRNA (perch, lake trout, herring from 20100318) Fragmentation

Fragmented mRNA according to Ambion’s Whole Transcriptome Sequencing Kit. Cleaned up sample using Ribominus Concentration Module (Invitrogen) according to Ambion’s WTS Analysis Kit. Samples were eluted w/20uL of H2O and stored @ -80C. Will Bioanalyze and speedvac at a later date.

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Bioanalyzer for SOLiD libraries – Total and mRNA from Perch, Lake Trout & Herring RNA samples (CONTINUED from yesterday)

Total and mRNA aliquots (~5ng/uL) were run on the Agilent Bioanalyzer Pico RNA chips.

Results:

The gel below shows the comparison/results of total RNA and subsequent mRNA isolations. The gel indicates the following:

  1. The HPWS09 total RNA (Herring) is totally degraded, but shows the expected profile in the mRNA prep. It would be extremely interesting to see if the degradation has any effect on sequencing, as the mRNA will get fragmented any way in the next step of library construction.

  2. mRNA isolations worked for all samples. Although one might be inclined to say that mRNA isolation did NOT work for the WB sample, one has to take in to consideration that the gel software adjusts the gel contrast to enhance low signals. That’s why all the mRNA samples exhibit a dark background. mRNA generates a broad, relatively weak signal when compared to a total RNA sample. So, the software attempts to boost the low signal for display purposes. Thus, if we were to decrease this signal boosting (or contrast) for the WB mRNA so that the background color matched the WB total RNA background color (white), the rRNA bands visible in the WB mRNA sample would fade to a point where they would not be visible. See the electropherogram overlay (below the gel) for a more visual comparison of this concept.

Electropherogram Overlays of WB total RNA and WB rRNA

The WB total RNA is the red graph which shows extremely high levels of rRNA (as expected). After subsequent mRNA isolation (the blue graph), the rRNA is virtually gone and no longer comprises a significant portion of the sample.

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