Illumina RNAseq Library Construction – 32 C.gigas Individuals

Took heat-fragmented RNA provided by Emma (see Emma’s Notebook, 7/3/2011) and proceeded to make first strand cDNA, as described in the Eli Meyer protocol for Illumina HiSeq. Master mix calcs are here. Samples were stored @ -20C after the reverse transcription and library construction will be continued tomorrow.

Oligo Reconstitution – Illumina RNAseq Library Oligos and Barcodes

Reconstituted all of the oligos and barcodes for library construction in TE (pH = 8.0) to a final concentration of 100uM. Created 10uM working stocks of all oligos and barcodes. All samples (stocks and working stocks) are stored @ -80C in their own box (Illumina Library Oligos & Barcodes) due to the fact that one of the oligos is an RNA oligo and requires storage at -80C.

Chloroform Clean Up – Lexie’s QPX RNA from 20110504

After submission of QPX samples to HTGU for Illumina library prep yesterday, I was notified that there was insufficient RNA for the QPX RNA samples. I checked the source RNA on the Roberts Lab NanoDrop1000 and determined that they had high phenol contamination (large peak at 270nm), which results in a large exaggeration in the OD260 absorbance (NanoDrop1000 report[JPEG]; notice terrible OD260/280 ratios; did not save screen shot of absorbance peaks.). As such, the concentrations that Lexie had listed in her notebook for these samples are highly inaccurate and highly inflated. To remove the phenol, I brought all of her QPX RNA samples from 20110504 up to ~200uL with 0.1%DEPC-H2O, added 200uL of chloroform, vortexed for 30s, spun at 12,500g RT for 15mins, and transferred aqueous phase to new tube. Then performed an ethanol precipitation on the aqueous phase. Added 0.1 vols of 3.0M sodium acetate (pH = 5.2), 2.5 vols of 100% EtOH, mixed and incubated at -20C for 1hr. Pelleted RNA by spinning at 16,000g 4C for 15mins.

Results:

As suspected, most of these samples have absolutely no RNA in them. However, the samples that do (the “Control” samples), look great! Pooled 2ug each of the RT Control a & b samples and pooled 2ug each of the 10C Control a & b samples (which are ATCC). Calculations are here. Will take them down to HTGU tomorrow to replace the bad samples that were provided yesterday.

qPCR – Detection of V.tubiashii Presence and Expression Using VtpA Primers in DNA/cDNA from yesterday

Ran qPCR with VtpA primers on cDNA and DNA (from yesterday) of C.gigas larvae to see levels of V.tubiashii compared to their water filter samples (see 20120326). Master mix calcs are here. Plate layout, cycling params, etc can be seen in the qPCR Report (see Results). Used 1uL of cDNA and 100ng (1uL) of DNA as template.

All samples were run in duplicate.

Results:

qPCR Data File (CFX96)
qPCR Report (PDF)

No detectable levels of expression (or, no expression at all) in any of the cDNA samples.

Below I’ve put together a very rough comparison of larvae levels, based off of the the standard curve. I have NOT done the full back calculations!! This is data straight out of the qPCR machine, using the standard curve. Due to the large range, I’ve graphed the data on a logarithmic scale so all the data is visible on the graph.

Reverse Transcription – DNased C.gigas Larval RNA from 20120427

Performed reverse transcription using random primers (Promega) diluted 1:100 (5ng/uL) with 175ng of DNased total RNA. Random primers were used because we will be targeting V.tubiashii RNA instead of eukaryotic RNA. Reverse transcription was performed with M-MLV Reverse Transcriptase (Promega) according to the manufacturer’s protocol. Calcs are here.

 

Created dilutions of all samples to 100ng/uL in a volume of 50uL in preparation for qPCR analysis. Calcs are here.

qPCR – Check DNased RNA from Earlier Today for Residual gDNA

Ran qPCR using V.tubiashii VtpA primers (from Elene; no SR ID). Used 0.5uL of each DNased RNA sample, which equals ~40ng of RNA, which would be the equivalent amount of RNA that would end up in a qPCR rxn after cDNA has been made (using 1uL of cDNA). Used the filter DNA extraction from samples #279 from DATE as a positive control. Master mix calcs are here. Plate layout, cycling params, etc. can be found in the qPCR Report (see Results).

Results:

qPCR Data File (CFX96)

qPCR Report (PDF)

All samples showed up negative, except for the positive control. Will proceed with making cDNA on Monday.