Tag Archives: Pacific herring

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.

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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.

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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.

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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.

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SOLiD ePCR – Herring fragmented cDNA library: 2LHKOD09

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

Processed herring fragmented cDNA library 2LHKOD09 (76.1.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) = 761pg/uL. Mixed 23.7uL of this diluted sample with 76.3uL 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 started and run O/N. The plate will be stored @ 4C until the two remaining libraries have been through ePCR. Then, all three libraries will be processed simulatneously.

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SOLiD Bead Titration – Herring fragmented cDNA library 3LHSITK09 (CONTINUED from ePCR yesterday)

Completed the remainder of the procedure for template bead titration, 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.

Templated bead count after breaking emulsion, in a 1:200 dilution: 110, 129, 115, 105, 143, 113. Average = 119.2 beads/square

Recovery: 119.2 beads/square x 10uL x 25 squares x 200 = 5.96×10^6 beads/uL

Total beads recovered: 5.96×10^6 beads/uL x 200uL = 1.19×10^9

Desired # of beads is between 1-2 billion. I recovered 1.19 billion. This is in the desired range.

Final count of enriched, templated-beads: 194, 181, 161, 214. Average = 187.5 beads/square

Final recovery: 187.5 beads/square x 10uL x 25 squares x 10 = 4.6875×10^5 beads/uL

Total enriched, templated beads recovered: 4.6875×10^5 beads/uL x 400uL = 1.875×10^8 beads

Enrichment efficiency percentage: 1.875×10^8 beads/1.19×10^9 x 100 = 15.8%

Beads were stored @ 4C until more templated beads are generated.

Results: This is excellent! Got desired recovery of beads after the ePCR and got an excellent yield of templated beads. Need 46 million for a section on an octet and got 188 million, plus the 12 million from yesterdays. These two samples will be pooled. Will now proceed with the remaining herring samples, using 1.5pM as the starting amount of template for each library.

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SOLiD ePCR – Herring fragmented cDNA library: 3LHSITK09

Due to low yield of templated beads (12×10^6) from the first run through (see SOLiD Bead Titration below), am repeating using 1.5pM of starting template (as opposed to 0.5pM used yesterday). It should be noted that there is NOT a linear relationship between the amount of starting template and the amount of enriched, templated beads one ends up with in this protocol. So, even though I am increasing the starting template by 3-fold, a 3-fold increase in the amount of enriched, templated beads is NOT expected (hopefully it’ll be more than that!).

Processed herring fragmented cDNA library 3LHSITK09 (88.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) = 885pg/uL. Mixed 20.3uL of this diluted sample with 79.7uL 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) in mid-December 2009. This oil phase is stable for 2 months @ 4C.

ePCR was started and run O/N. The rest of the procedure will be finished tomorrow.

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SOLiD Bead Titration – Herring fragmented cDNA library 3LHSITK09(CONTINUED from ePCR yesterday)

Completed the remainder of the procedure for template bead titration, according to the ABI “Templated Bead Preparation Guide” following the “full-scale” protocol.

Templated bead recovery after breaking emulsion, in a 1:200 dilution: 92, 105, 97, 99, 89, 96. Average = 96.3 beads/square

Calculation of beads: Avg (beads/square) x Volume in hemocytometer (uL) x total # squares on hemocytometer (squares) x dilution factor = beads/uL

Total bead recovery: beads/uL x volume of beads (uL)

So, my recovery is: 96.3 beads/square x 10uL x 25 squares x 200 = 4.817×10^6 beads/uL

Total beads recovered: 4.817×10^6 beads/uL x 200uL = 9.63×10^8 beads.

Desired # of beads is between 1-2 billion. I recovered 963 million. Close.

Final count of enriched, templated-beads: 8, 14, 16, 10. Average = 12

My final recovery is: 12 beads/square x 10uL x 25 sqaures x 10 = 30000 beads/uL.

Total enriched, templated beads recovered: 30000 beads/uL x 400uL = 12×10^6 beads.

Enrichment efficiency percentage calculation: (# templated beads)/(Starting # beads) x 100

My enrichment efficiency percentage: 12×10^6 beads/9.63×10^8 beads x 100 = 1.2%

Beads were stored @ 4C until more templated beads are generated.

Results: The yield of beads is not entirely unexpected, according to Rhonda, because I started the procedure using only 0.5pM of the library. However, each section on an octet slide (which is what we plan on using) requires 46 million beads. Clearly this is short of that quantity. The procedure will be repeated with a greater amount of starting cDNA library. It should be noted that there is NOT a linear relationship between the amount of starting template and the amount of enriched, templated beads one ends up with in this protocol. So, even though I will be increasing the starting amount of template by 3-fold, a 3-fold increase in the amount of enriched, templated beads is NOT expected (hopefully it’ll be more than that!).

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SOLiD ePCR – Herring fragmented cDNA library: 3LHSITK09 (from 20091209)

Processed herring fragmented cDNA library 3LHSITK09 (88.ng/uL) according to the ABI “Templated Bead Preparation Guide” following the “full-scale” protocol. Made a 1:1000 dilution (1uL library, 999uL 1x Low TE) = 88.5pg/uL. Mixed 67.8uL of this diluted sample with 32.2uL 1x Low TE to get a final concentration of 60pg/uL (500pM, according to ABI protocol). Oil phase used was previously prepared by Jesse (Seeb lab) in mid-December 2009. This oil phase is stable for 2 months @ 4C.

ePCR was started (Rhonda will put plate in fridge for storage O/N) and the rest of the procedure will be finished tomorrow.

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Bioanalyzer – Herring Liver cDNA for SOLiD Libraries

Samples were run on the DNA 1000 chip for cDNA smear analysis.

Results: 2 of the 4 samples (2L & 3L) look perfect (<20% of cDNA in the 25-150bp range). 6L has <20% of the cDNA in the 25-150bp range (which is perfect), but exhibits a “smear” from ~250-500bp. cDNA in this range suggests overamplification, which will skew gene expression profile. Can repeat PCR for 6L using outer gel slices and reduce the number of cycles to prevent overamplification, if desired. Spoke to Steven and since these samples won’t be used to evaluate gene expression (they’re for SNP discovery), we won’t worry about it for the time being.

Sample 4L has some very strange signals being generated in the ~500-800bp range. Additionally, the virtual gel image (not shown) shows a great deal of smearing, unlike the other samples.

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