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.
Tag Archives: SOLiD libraries
mRNA Precipitation – Herring Liver mRNA for SOLiD Libraries (continued from yesterday)
Spun samples 16,000g, 30mins, 4C. Discarded supe, quick spun tubes, removed residual supe, washed with 1mL 70% EtOH. Spun samples 16,000g, 15mins, 4C. Discarded supe, quick spun tubes, removed residual supe, resuspended in 8.5uL of nuclease-free H2O. Stored @ -80C until ready to proceed with fragmentation for SOLiD libraries.
RNA Precipitation – Herring Liver RNA for SOLiD Libraries (continued from yesterday)
RNA Precipitation
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.
mRNA Isolation
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.
Results:
Yields:
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
mRNA Precipitation
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.
RNA Precipitation – Herring Liver RNA for SOLiD Libraries
50uL of RNA from each of the following were precipitated O/N @ -20C:
2L HKOD09 – 2.157 ug/uL x 50uL = 107.85ug
4L HTOG09 – 2.089 ug/uL x 50uL = 104.45ug
3L HSITK09 – 1.089 ug/uL x 50uL = 54.45ug
6L HPWS09 – 1.703 ug/uL x 50uL = 85.15ug
0.1 volumes (5uL) of 3M NaOAc ph = 5.2 was added to each sample. Then 2 volumes (110uL) of 100% EtOH was added. Samples were vortexed and incubated O/N @ -20C.
Bioanalyzer Submission – Trout RBC, Colleen’s gigas GE sample, Mac’s DH/BB PCR for SOLiD WTK
Samples were delivered for analysis on the DNA 1000 chip.
UPDATE 20091008 They do not have the DNA 1000 kit in stock. Will be using High Sensitivity Kit instead. Will have in stock on Tuesday.
Results:
Reverse Transcription/cDNA purification/Emulsion PCR – Ligation rxns of trout fragmented RNA for SOLiD WTK (from yesterday)
The four samples from yesterday were prepared according to the Agilent SOLiD WTK protocol. Briefly:
Results: All four samples appear to have cDNA. Interestingly, the “Amped cDNA trout RBC control ribo(-)” sample was the sample that had no detectable RNA after fragmentation, BUT this sample produced the highest yield of cDNA… See below.
1.5uL of each sample was transferred to a 0.5mL snap cap tube and stored @ -80C in the “Samples for Bioanalyzer” box for submission on the DNA 1000 Chip.
The Yellow/Brown plot above is the “Amped cDNA trout RBC poly I:C ribo(-) & polyA” sample and exhibits a strange profile at the 220-230nm range that differs than the three other samples.
Adapter Ligation – Rick’s trout fragmented control/poly I:C samples for SOLiD WTK
See the Next Gen Seq Library Database for more info. Processed the 4 samples (one set Ribominus only, one set Ribominus + PolyA enriched) according to the Agilent WTK. Briefly:
- Speedvac’d samples to dryness
- Resuspended RNA in 3uL H2O
- Adapter rxn. Used all 3uL of RNA (used only 1uL of RBC Ribo only sample due to high concentration)
- Ligation rxn
Incubated 16C for 16hrs.
Bioanalyzer Submission – Rick’s trout RBC samples (various dates)
Submitted Rick’s trout RBC samples to FHRC for bioanalysis using the PicoChip for use with the SOLiD WTK. Submission sheet is here.
Results: Received 20091001.
Lanes 1 & 2 = ribo-depleted AND polyA enriched
Lanes 3 & 4 = ribo-depleted only
Lanes 5 & 6 = total RNA
Lanes 7 & 8 = ribo-depleted only
RNA Fragmentation – Rick’s trout RBC samples prepped earlier today
EtOH Precipitaiton – Rick’s trout Ribosomoal-depleted RNA for SOLiD WTK (continued from yesterday)
Continued precipitation. Spun samples 30 mins, 16,000g, 4C. Removed supe. Added 1mL 70% EtOH. Spun samples 15mins, 16,000g, 4C. Removed supe. Resuspended in 8uL H2O. Proceeded with SOLiD WTK fragmentation.
RNA Fragmentation
Samples were fragmented according to the Whole Transcriptome Kit protocol. Samples were then cleaned up using Invitrogen’s RiboMinus Concentration Module, according to SOLiD WTK protocol. Briefly:
- Added 1X volume of binding buffer (100uL)
- Added 100% EtOH (250uL)
- Eluted with 20uL of H2O.
Samples were spec’d.
Results:
Control Sample – Virtually nothing there. Hopefully it’s just too dilute for the NanoDrop, however I have a feeling this sample is bad (degraded?) 1.5uL of the sample has been transferred to a 0.5mL snap cap tube to send off for the Bioanalyzer.
Poly I:C Sample – Looks great, excellent recovery. 0.25uL of this sample was transferred to a 0.5mL snap cap tube containing 1.25uL of H2O to send off for the Bioanalyzer.
Samples were stored @ 80C until resutls from the Bioanalyzer are received.
EtOH Precipitation – Rick’s trout Ribosomoal-depleted RNA for SOLiD WTK (from today)
The “control” and “poly I:C” samples prepared earlier today were EtOH precipitated in preparation for fragmentation.
Added the following to each sample:
- 18uL 5M ammonium acetate
- 1uL glycogen
- 2 vols. of 100% EtOH (74uL)
Samples were incubated O/N @ -80C.