mRNA was isolated for SOLiD sequencing by HTGU. Made two pools of San Nick RNA (Control and Exposed) using equal amounts (5ug) of each individual sample. Individual samples used can be found here. mRNA was isolated using Ambion’s Micro PolyAPurist Kit according to protocol. Procedure was performed two times on each pool and then EtOH precipitated. Samples were resuspended in 10uL of The RNA Storage Solution provided in the kit and spec’d on the Roberts Lab ND1000. Samples were stored @ -80C in the “Next Gen Sequencing Libraries” box.
Yields are pretty good from both samples (~500ng). However, the OD260/280 values are rather poor.
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.
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.
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.
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.
Total and mRNA aliquots (~5ng/uL) were run on the Agilent Bioanalyzer Pico RNA chips.
The gel below shows the comparison/results of total RNA and subsequent mRNA isolations. The gel indicates the following:
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.
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.
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.
Samples from 20091203. 0.5uL was removed from each and transferred to separate tubes and diluted to < 5ng/uL for subsequent Bioanalyzer analysis using the Pico chip. Samples were fragmented using RNase III according to the Ambion WTK protocol and then cleaned up/concentrated using the Invitrogen RiboMinus Concentration Module according to the Ambion WTK protocol.
Samples were spec’d prior to running on the Bioanalyzer:
Concentrations/absorbance values are not accurate when using the NanaDrop after using the RiboMinus Concentration module, according to the Ambion WTK protocol. However, yields seem pretty good…
Total, mRNA and fragmented mRNA from each of the four samples was run on the Pico chip with the Eukaryote Total RNA Bioanalyzer protocol.
The 2L tot (total RNA) and 3L tot (total RNA) samples are clearly very good quality. 2L tot does exhibit some very slight degradation, though. 4L tot (total RNA) and 6L tot (total RNA) show a much greater degree of degradation. All mRNA samples show complete removal of any trace, contaminating rRNA. The fragmented samples (the last four samples on the gel image above) all appear to be perfect. The 4L frag sample simply has less RNA loaded and that is why it is not as dark as the other three fragmented samples. Despite the degradation in the 4L tot and 6L tot samples, the fragmentation profile looks good and we will proceed with making the cDNA libraries for those samples.
Samples were pelleted for 30mins, 16,000g @ 4C. Supe was discarded, samples quick spun, residual supe removed and then washed with 1mL 70% EtOH. Tubes were vortexed until pellet came off of bottom of tube and then spun 15mins, 16,000g @ 4C. Supe was discarded, samples quick spun, residual supe removed and then resuspended in 250uL of 0.1% DEPC-H2O in preparation for mRNA isolation using the Ambion Micro PolyA Purist Kit.
Clean RNA from earlier today was processed according to the Ambion Micro PolyA Purist Kit to isolate mRNA. This procedure was done two times to ensure full mRNA enrichment of the samples. Samples were then spec’d.
3L – 10.66 ng/uL x 200uL = 2.132ug
6L – 6.97 ng/uL x 200uL = 1.394ug
2L – 10.89 ng/uL x 200uL = 2.178ug
4L – 6.43 ng/uL x 200uL = 1.286ug
Precipitation of mRNA from earlier today in preparation for fragmentation. Fragmentation requires mRNA volumes of <8uL, so after precipitation I will resuspend pellets in 8uL of 0.1% DEPC-H2O. Will use 1ug (this means 1/2 of 3L and 2L & all of 6L and 4L) of mRNA from each sample for precipitation. Remainder of 3L and 2L samples were stored @ -80C in “Herring RNA Box #1.”Samples were precipitated by adding 0.1 volumes 5M ammonium acetate, 1uL glycogen and 2 volumes of 100% EtOH. Samples were incubated O/N @ -20C.