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.
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.
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.
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:
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 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.
Data from MoGene was received today on two DVDs and one HDD. Data is two runs of two libraries, due to MoGene concerns that the data of the first run looked bad (too few reads). They performed a second run at no charge and provided us with that data as well.
Data is located here: http://owl.fish.washington.edu/nightingales/C_pallasii/