The overall goal of this procedure is to assay the gene regulatory activities of thousands of DNA sequences in parallel. This is accomplished by first constructing a reporter library where the DNA sequences of interest are linked to reporter genes with one or more identifying sequence tags in their three prime ur. The second step of the procedure is to transfect this reporter library into a population of cells.
The final step is to count the number of occurrences of each of the identifying sequence tags in the reporter mRNAs and the corresponding reporter library by deep sequencing. Ultimately, results can be used to infer the relationship between the DNA sequence and activities of gene regulatory elements through computational analysis of the sequence tag counts. The main advantage of this technique over existing methods like luciferase or GFP based reporter assays, is that it allows you to assay thousands of regulatory sequences in a single dish of cultured cells.
This method can help answer key questions in the gene regulation field, such as which nucleotides within the promoter or enhancer essential based activity. This method can be used for a wide variety of other experiment design, such as screening large library of putative regulator elements for activity in a specific cellular states. Demonstrating the reporter library construction procedure will be Peter Rugo, a senior research associate from biotechnology labs, demonstrating the reporter library TRANSFECT and RNA recovery will be Lee Wang, a senior research associate from my group, Massively parallel reporter assays henceforth refer to as NPRA, begin with the design and synthesis of sequences to be assayed for regulatory activity.
And tag denotes one or more identifying tags. KPN one and XBA one restriction sites are situated in between. Re suspend the synthesized oligonucleotide library in 100 microliters of TE 0.1 buffer.
Run the oligonucleotide library on a 10%TBE urea denaturing poly acrylamide gel. Load about one ole in each lane and analyze the gel with a single stranded DNA sensitive fluorescent stain. If there is a discreet ban corresponding to the full length oligonucleotides, then cut it out and elute the oligos into 100 microliters of TE 0.1 overnight at room temperature and with shaking using the gel purified or raw oligonucleotide library.
Set up an emulsion PCR to amplify the library and add SI one tails. Set up 50 microliter reactions as described in table two. Then combine the 50 microliter reaction mix with 300 microliters of oil and surfactant mixture from the my DNA emulsion and purification kit for five minutes at four degrees Celsius vortexes mix into an emulsion.
Then distribute the emulsion into PCR tubes or plates as 50 microliter reactions and perform 20 cycles of PCR after the PCR. Pull the 350 microliter emulsions back together. Then add one milliliter of Isobutanol and vortex to mixture.
Briefly purify the amplification product using a DNA purification column and run an Eloqua on a 2%or 4%agros gel to assess its quality. The serum amplification of a complex oil libraries can introduce representation bias, RIC Applicon and other artifacts. It is essential to minimize the number of PCR cycles used for additional quality control.
It is recommended to do full sequencing library before proceeding to the next steps To prepare the linearized plasmid backbone. First digestive P pra one vector with SFI one at 50 degrees Celsius for two hours. Then run the digested products out on a 1%gel and collect the DNA from the 2.5 KILOBASE backbone band using a gel purification spin column.
Next, digest the emulsion PCR products in the same way and collect the products with the DNA purification column as well. Now set up a ligation reaction with 100 nanograms of digested PCR and 50 nanograms of linearized plasmid. Use T four ligase and run the reaction overnight at 16 degrees Celsius the next day.
Inactivate the ligation reaction by heating it at 65 degrees Celsius for 20 minutes. Then transform two microliter aliquots of the ligation reaction products into 50 microliter aliquots of electro competent equali. Recover the cells in 800 microliters of SOC medium at 37 degrees Celsius with shaking for one hour.
Then pool all the parallel transformations and evaluate them by plating serial dilutions onto LB agar with 50 to a hundred micrograms of carbon penicillin per milliliter. Incubate the plates overnight at 37 degrees Celsius and estimate the total CFU the next day. Immediately after transformation, use the remainder of the transformed cells to inoculate 200 milliliters of LB supplemented with 100 micrograms of carbonic per milliliter.
Grow the cells at 37 degrees Celsius overnight and with shaking the following day, isolate the plasma DNA following standard procedures to confirm the presence of inserts. Digest an aliquot of the isolated library with SFI one and run out the products on a 1%aros gel Linearize the plasmid library with serial digestions using KPN one and XBA one first digest with KPN one at 37 degrees Celsius for an hour and purify the products using magnetic beads. Second, digest a products with XBA one plus a unit of shrimp alkaline phosphatase at 37 degrees Celsius for two hours later.
Inactivate the reaction at 65 degrees Celsius for five minutes, and then purify the products using magnetic beads. Run an aliquot on a 1%gel, and if any uncut plasmid is visible, gel purify the linearized fragment now to generate an NPRA library suitable for transfection into mammalian cells. First, prepare a compatible luck to open reading frame fragment from P-M-P-R-A donor one.
Digest this plasmid with KPN one and XBA one at 37 degrees Celsius for an hour. Then run the products out on a 1%AGROS gel. Excise the open reading frame fragment in this case at 1.7 kilobases and purify using a gel purification spin column.
Then ligate the linearized open reading frame into the Linearized intermediate library as described for the previous ligation. Transform the ligation products into e coli cells and isolate final NPRA plasmid library as previously described. If you're not careful, carry over.
The adjusted plasmids and subplated products can greatly reduce the proportion of correct constructs in final plasmid pool. Be sure to isolate the correct band and check for background before proceeding to the next steps. As a check digest an Eloqua of one or two micrograms of the NPRA library with KPN one.
Run this out on a 1%gel and if it runs as a single band, proceed. Otherwise, consult the text protocol on how to deal with contamination to transfect cultured cells. With the NPRA.
Plasmids conditions must be optimized for each cell type. For example, 5 million HK 2 93 T 17 cells grown to about 50%confluence in a 10 centimeter culture dish can be transfected with 10 micrograms of plasmid DNA in one milliliter of ofm, one reduced serum medium using 30 microliters of lipo LTX and 10 microliters of plus reagent. In this case, remove the transfection mixture about five hours and allow the cells to recover for one or two days later, harvest the cells and use a kit to isolate the poly positive mRNA.
Then make a tag, seek sequencing library and analyze the results. NPRA facilitates high resolution quantitative dissection of the sequence activity relationships of transcriptional regulatory elements. A successful NPRA experiment typically yields highly reproducible measurements for the majority of sequences in the Transfected library.
If reproducibility is poor, the concentration of reporter mRNAs in the recovered RNA is probably too low. This can happen because of low absolute activity among the assay sequences or because of low transfection efficiency. In these successful n PRAs, 37, 000 variants of a 1 45 base pair sequence upstream of the human interferon beta gene were analyzed with or without exposure to sendi virus.
The promoter Tata box and known proximal enhancer can be clearly identified as information-rich regions in a virus dependent manner. Once mastered, this technique can be done in about a week if it is performed properly Following this procedure. Other methods like EMCE and protein DNA affinity capture can be performed in order to answer additional questions like which transcription factor is binding to critical nucleotides within an active DNA sequence.
After watching this video, you should have a good understanding of how to prepare reporter libraries to assay the gene regulatory activities of thousands of DNA sequences in parallel.
