Tag Archives: Carmel

Templated Bead Prep SOLiD Libraries – Abalone CC, CE pools and yellow perch CT, PQ libraries

All libraries were prepped according to ABI’s “full-scale” bead prep protocol. Initial bead counts were performed using a hemocytometer in a 1:200 dilution:

Formula for calculating bead counts:

Average hemo count x hemo volume x hemo squares x dilution x bead volume

Initial Bead Counts

CC: 127, 120, 126, 113. Average = 96 Count: 96 x 10 x 25 x 200 x 200 = 9.6 x 10^8 beads

CE: 99, 93, 115, 102. Average = 102.25 Count: 102.25 x 10 x 25 x 200 x 200 = 1.0225 x 10^9 beads

CT: 118, 113, 109, 111. Average = 112.75 Count: 112.75 x 10 x 25 x 200 x 200 = 1.1275 x 10^9 beads

PQ: 109, 116, 111, 86. Average = 105.5 Count: 105.5 x 10 x 25 x 200 x 200 = 1.055 x 10^9 beads

Templated Bead Counts

Templated bead counts were performed using a hemocytometer with a 1:10 dilution:

CC: 217, 226, 208, 219 Average = 217.5 Count: 217.5 x 10 x 25 x 10 x 400 = 2.175 x 10^8 beads

CE: 211, 169, 162, 180 Average = 180.5 Count: 180.5 x 10 x 25 x 10 x 400 = 1.805 x 10^8 beads

CT: 223, 219, 254, 214 Average = 227.5 Count: 227.5 x 10 x 25 x 10 x 400 = 2.275 x 10^8 beads

PQ: 176, 177, 161, 163 Average = 169.25 Count: 169.25 x 10 x 25 x 10 x 400 = 1.6925 x 10^8 beads

Templated Bead Recovery: Final bead count divided by initial bead count x 100 = % recovery

CC = 2.17 x 10^8 beads/9.6 x 10^8 beads x 100 = 22.7%

CE = 1.805 x10^8 beads/10.225 x 10^8 beads x 100 = 17.7%

CT = 2.275 x 10^8 beads/11.275 x 10^8 beads x 100 = 20.2%

PQ = 1.6925 x 10^8 beads/10.55 x 10^8 beads x 100 = 16.04%

Results: Yields of templated beads look fabulous. Recoveries of templated beads are a bit on the high side (desired recoveries are between 5-15%, with 20% being the “cutoff” that Rhonda’s lab uses for runs. The CC and CT samples cross this cutoff value. Will consult with Steven to see what how he wants to proceed (i.e. new ePCRs?). Beads stored @ 4C until ready for running on the SOLiD.

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ePCR SOLiD Libraries – Abalone CC, CE pools and yellow perch CT SOLiD libraries (from 20100408)

Emulsion PCR (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:

CC (13.8ng/uL): 1.3uL in 100uL

CE (23.01ng/uL): 1.56uL in 200uL

CT (63.8ng/uL): 1.41 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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RNA Precipitation and Fragmentation for SOLiD Libraries – Pooled abalone mRNA (from yesterday)

mRNA was precipitated according to Ambion’s MicroPolyA Purist Kit protocol. Added 0.1vols of ammonium acetate, 2.5vols of 100% EtOH and incubated 30mins @ -80C. Samples were pelleted, washed with 1mL 70% EtOH, pelleted, resuspended in 8uL of nuclease-free H2O and spec’d:

After precipitation, samples were fragmented with RNase III according to the Ambion Whole Transcriptome Analysis Kit protocol and then cleaned up using the Invitrogen Ribominus Concentration Module, according to the Ambion Whole Transcriptome Analysis Kit protocol. 0.5uL of each sample was removed for analysis on the Bioanalyzer.

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RNA Precipitation & mRNA Isolation for SOLiD Libraries – Pooled abalone total RNA: Carmel control, Carmel exposed

RNA of 8 samples from each group was pooled equally from each individual. RNA was precipitated according to Ambion’s MicroPolyA Purist Kit. Used 0.1 volumes of 3M NaAOc, pH=5.2, 2.5vols of 100% EtOH and incubated 30min @ -80C. Pelleted RNA 16,000g, 30mins. Washed pellet w/70% EtOH and pelleted RNA 16,000g, 15mins. Pellets were resuspended in 50uL nuclease-free H2O and spec’d:

Total RNA pools look really nice. ~45ug of total RNA in each sample.

Isolated mRNA from each pool using Ambion’s MicroPolyA Purist Kit according to protocol. Samples were processed 2x as recommended by Ambion’s SOLiD Whole Transcriptome Analysis Kit. Final elution was 200uL of The RNA Storage Solution. Samples were spec’d:

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