Tag Archives: library prep

Bioanalyzer Total, mRNA and post-fragmentation SOLiD Libraries – Abalone pools

0.5uL of fragmented mRNA from each library (combined with 0.5uL) was run on Agilent Bioanalyzer 2100 using RNA Pico chips/reagents according to Agilent’s protocol.

Results:

Total RNA shows a single, distinct rRNA band, along with some low-molecular weight RNA (i.e. degraded) in both total RNA samples. mRNA samples exhibit the expected “smear” that spans a large range of molecular weights. Both mRNA samples also show residual rRNA bands, but their concentrations should be extremely low. Fragmented samples show the expected strong band of low-molecular weight RNA. The CE frag sample exhibits some larger banding, which is probably background signal (compare to the empty lane labelled “Sample 7″).

Will proceed with rest of library procedure for both fragmented samples.

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.

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:

Bioanalyzer for SOLiD Libraries – Fragmented mRNA from Perch, Lake Trout & Herring RNA samples

1uL of each sample from 20100325 was run on the Agilent 2100 Bioanalyzer on a RNA Pico 6000 chip to evaluate RNA quantity and fragmentation.

Results:

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.

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.

mRNA Precipitation for SOLiD – Perch, Lake Trout, & Herring mRNA (CONTINUED from yesterday)

mRNA was pelleted and washed according to Ambion’s MicroPolyA Purist Kit. Pellets were resuspended in 8uL nuclease-free H2O and spec’d. 0.5uL was taken from each sample, transferred to a fresh tube, diluted to ~5ng/uL and stored @ -80C for eventual Bioanalyzer analysis. mRNA samples were stored @ -80C until we receive the Ribominus Concentration Module Kit from Invitrogen (turns out we didn’t have any!) for cleaning up the RNA after fragmentation.

Results:

Overall, this mRNA doesn’t look that great. However, I did notice that all samples had (to varying degrees) particulate matter that wouldn’t dissolve. Prior to spec’ing, the particulate matter was pelleted so as to not interfere. All samples will continue to be prepped for SOLiD analysis despite poor 260/280 ratios and low yields.

mRNA Isolation for SOLiD – Perch, Lake Trout, and Herring total RNA

Received pooled lean and siscowet RNA from Rick. Samples will be processed immediately for SOLiD fragment libraries. Two 1.5mL snap cap tubes labelled:

L.T. 2ug muscle sisco pool

L.T. 2ug muscle lean pool

RNA was first precipitated according to the Ambion MicroPolyA Purist Kit protocol (0.1 vol 5M ammonium acetate, 1uL glycogen, 2.5 vols 100% EtOH). Samples were incubated @ -80C for 30mins. Samples were resusupended in 250uL nuclease-free H2O and spec’d.

 

Starting quantities PRIOR to total RNA precipitation:

Perch Samples:

WB tRNA (WB tRNA ~12ug 24.5uL)

PQ tRNA (PW tRNA ~12ug 21.88uL)

CT tRNA (CT tRNA ~12ug 25uL)

Herring:

1 G/O HPWS09 (20ug)

Lake Trout:

L.T. 20ug muscle sisco pool

L.T. 20ug muscle lean pool

Removed 1uL of each sample, diluted to ~5ng/uL and stored @ -80C to run on the Bioanalyzer.

Used Ambion MicroPolyA Purist Kit according to protocol. Samples were treated twice to ensure elimination of rRNA from the samples. After second run through MicroPolyA Purist, samples were EtOH precipitated O/N @ -20C according to Ambion’s MicroPolyA Purist protocol.

SOLiD Bead Titration – Herring fragmented cDNA libraries: 2LHKOD09, 4LHTOG09, 6LHPWS09

Continued with templated bead prep from ePCRs for these libraries. Samples were processed according to the ABI “Templated Bead Preparation Guide” following the “full-scale” protocol.

To see explanations of the various calculations below, see the “SOLiD Bead Titration – Herring fragmented cDNA library 3LHSITK09 (CONTINUED from ePCR yesterday)” from 20100107.

2LHKOD09:

Initial counts: 109, 129, 112, 115 —- Avg. = 116.25 beads/square

Beads: 116.25 x 10 x 25 x 200 = 5.8125×10^6 beads/uL x 200uL = 1.1625×10^9 beads

Templated beads counts: 205, 199, 197, 210 —– Avg. = 210 beads/square

Templated beads: 210 x 10 x 25 x 10 = 5.06875×10^5 beads/uL x 400uL = 2.0275×10^8 beads

Efficiency: 17.44%

4LHTOG09:

Initial counts: 124, 124, 117, 104 —– Avg. = 117.25 beads/sqaure

Beads: 117.25 x 10 x 25 x 200 = 5.8625×10^6 beads/uL x 200uL = 1.1725×10^9 beads

Templated beads counts: 139, 135, 145, 140 —- Avg. = 139.75 beads/square

Templated beads: 139.75 x 10 x 25 x 10 = 3.49375×10^5 beads/uL x 400uL = 1.3975×10^8 beads

Efficiency: 11.92%

6LHPWS09:

Initial counts: 135, 106, 123, 124 —- Avg. = 122 beads/square

Beads: 122 x 10 x 25 x 200 = 6.1×10^6 beads/uL x 200uL = 1.22×10^9 beads

Templated beads counts: 141, 171, 164, 170 —– Avg. = 161.5 beads/square

Templated beads: 161.5 x 10 x 25 x 10 = 4.0375×10^5 beads/uL x 400uL = 1.615×10^8 beads

Efficiency: 13.24%

All beads were stored @ 4C until ready for bead deposition and work flow analysis run.

Results:

Rhonda Morales (from Ginger’s lab who is responsible for running/maintaining the SOLiD at the CEG) says the numbers on all samples look perfect! Will proceed to work flow analysis once Jesse’s samples are ready (ETA of Jan. 27th, 2010).

Here are reagent lot numbers for the Bead Titration.

SOLiD ePCRs – Herring cDNA libraries

Herring fragmented cDNA library: 4LHTOG09

Using 1.5pM of starting template, based on success of the 3LHSITK09 bead prep (see 20100108).

Processed herring fragmented cDNA library 4LHTOG09 (20.1.ng/uL) according to the ABI “Templated Bead Preparation Guide” following the “full-scale” protocol. Made a 1:100 dilution (1uL library, 99uL 1x Low TE) = 201pg/uL. Mixed 89.6uL of this diluted sample with 10.4uL 1x Low TE to get a final concentration of 180pg/uL (1.5pM, according to ABI protocol). Oil phase used was previously prepared by Jesse (Seeb lab) 1/11/2010. This oil phase is stable for 2 months @ 4C.

ePCR was run. The plate will be stored @ 4C until the two remaining libraries have been through ePCR. Then, all three libraries will be processed simulatneously.

Herring fragmented cDNA library: 6LHPWS09

Using 1.5pM of starting template, based on success of the 3LHSITK09 bead prep (see 20100108).

Processed herring fragmented cDNA library 6LHPWS09 (51.4.ng/uL) according to the ABI “Templated Bead Preparation Guide” following the “full-scale” protocol. Made a 1:100 dilution (1uL library, 99uL 1x Low TE) = 541pg/uL. Mixed 35uL of this diluted sample with 65uL 1x Low TE to get a final concentration of 180pg/uL (1.5pM, according to ABI protocol). Oil phase used was previously prepared by Jesse (Seeb lab) 1/11/2010. This oil phase is stable for 2 months @ 4C.

ePCR was run. The plate will be stored @ 4C until the two remaining libraries have been through ePCR. Then, all three libraries will be processed simultaneously.