Tag Archives: Whole Transcriptome Analysis Kit

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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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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Emulsion PCR – Herring Liver cDNA for SOLiD Libraries

Emulsion PCR was performed with the two inner gel bands cut out earlier today according to the Ambio WTK protocol. PCR was run for 15 cycles. After the PCR, the samples were cleaned up using the Invitrogen PureLink PCR Micro Kit, according to the Ambion WTK protocol. The cleaned up cDNA (referred to as “libraries” from now on) was spec’d prior to running on the Bioanalyzer.

Results:

All samples look good EXCEPT for 4L. It has a terrible 260/230 ratio and has a very low concentration, relative to the other three samples. Although not pictured here, the absorbance curve of the 4L sample was extremely poor and broad, unlike the other three samples.

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Reverse Transcription – Herring Liver mRNA for SOLiD Libraries

Ligation reactions from yesterday were subject to reverse transcription according to protocol. Master mix workup info is here. Samples were incubated @ 42C, 30mins and then cleaned up using the Qiagen MiniElute PCR Purification Kit, according to the Ambion WTK protocol.

After RT rxn, samples were run on a Novex 6% TBE-Urea gel according to Ambion WTK protocol. Samples were loaded, left to right: 2L, 3L, 4L, 6L. Ladders are to the left of each sample. The break in the smear in the 6L sample is a tear in the gel.

The recommended range of cDNA (100-200bp) were excised from the gel and were cut into four pieces, according to the Ambion WTK protocol. The two outer gel slices from each sample will be stored @-20C. Then proceeded to the emulsion PCR. Here is an image of the gel after the cDNA was cut out:

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RNA Adapter Hybridization and Ligation – Herring Liver mRNA for SOLiD Libraries

RNA from yesterday was speedvac’d to dryness and resuspended in 3uL of nuclease-free H2O. Samples were mixed with Adaptor Mix A and hybridized according to Ambion WTK protocol. Samples were then ligated for 16hrs @ 16C, according to Ambion WTK protocol.

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