The overall goal of this procedure is to generate Illumina mRNA sequence libraries from small amounts of starting material. The first step of the procedure is to reverse transcribe total RNA with DTT seven primers to produce CD NA.The cd NA is used for RNA amplification by in vitro transcription. Subsequently, the amplified RNA is subjected to random fragmentation.
Reverse transcription of the fragmented RNA produces CD NA, which undergoes adapter ligation size selection, and PCR amplification. Ultimately, this protocol shows the gene expression profile of the sample through measurement of gene copy numbers. The main advantage of this technique is that it requires only 10 nanograms of starting total RNA.
That is orders of magnitude less than what is required from other existing protocols. Moreover, it can generate paired in libraries from such small starting RNA quantities. This method will allow scientists to explore novel questions in biology by the, allowing the determination of gene expression profiles from difficult to obtain cell populations such as those derived from laser capture, micro dissection, or from flow Cytometry.
We first have the idea developing this method when we needed to profile gene expression from a very limited number of cells differentiated from human amnionic stem cells. Visual demonstration of this method is critical as the RNA handling steps are difficult to learn due to the very small amount of RNA that can be easily degraded. Demonstrating the procedure will be Angela Elwell, Jennifer Bolen, and Baal Kimm Wynn from my laboratory.
The method for preparing both single read and paired end sequencing libraries in parallel is a multi-step procedure as shown in this flow chart. First, total RNA is isolated from the cells or tissues of interest and used to prepare double stranded CD NA Polyadenylated messenger RNAs are then selectively amplified from the CD NA using the T seven based ewin linear amplification method. Next, the amplified poly a A is randomly fragmented.
After the fragmented RNA is cleaned up, it is reversed transcribed to CD NA.The CD NA is end repaired and a bases are added to the three prime end of the DNA fragments. After that, the DNA is adapter ligated and not one digestion is carried out. In the case of the single Reed Library, the DNA library is then isolated by gel purification and amplified by PCR to ligate the Illumina adapters.
Finally, after the DNA library is quantitated, it is ready for sequencing. For the purposes of this video, only selected procedures within this protocol will be demonstrated. Reverse transcription of the fragmented polyA mRNAs to cDNA involves first strand and second strand synthesis.
For first strand synthesis for the single read library, add the following reagents in A PCR tube, 10 microliters of fragmented poly mRNA and one microliter of not one random nonamer primer. The sequence of the not one nonamer primer is shown here. The five prime proximal sequence shown in green is the not one restriction site.
While the sequence shown in blue is the reverse compliment of illumina's adapter B sequence from the chip sequence kit incubate the mixture at 70 degrees Celsius for five minutes in the thermocycler and then quickly chill on ice. While keeping the mixture on ice, add these reagents from the superscript three kit four microliters of five x first strand buffer, two microliters of DTT and 1.5 microliters of DNTP mixture In the DNTP mixture, use five methyl DCTP instead of DCTP. Put the tube in the thermocycler at 42 degrees Celsius for two minutes and then add one microliter of superscript three, reverse transcriptase and incubate at 42 degrees Celsius for one hour.
Next, set up the reaction for first strand synthesis for the para end library. Add the following reagents in A PCR tube, 10 microliters of fragmented poly NNA and one microliter of random hexamer primer. Incubate at 65 degrees Celsius for five minutes in the thermocycler and then quickly chill on ice.
Keeping the tube on ice, add the following reagents from the superscript three First strand kit four microliters of five x first strand buffer, two microliters of DTT and 1.5 liters of DN NTPs. Incubate in the thermocycler at 45 degrees Celsius for one minute. Then add one microliter of superscript three, reverse transcriptase and incubate at 45 degrees Celsius for one hour after one hour.
Both reactions are ready for second strand synthesis. Add to each of the mixtures on ice, these reagents from the superscript two kit 91 microliters of free water, 30 microliters of five x second strand buffer. Three microliters of DN NTPs, one microliter of e coli, DNA Ligase, four microliters of e coli, DNA polymerase and one microliter of E coli R nase H.Mix each tube by inversion, then spin briefly and incubate at 16 degrees Celsius for two hours.
For end repair of the CDNA for the single read library, add to 40 microliters of purified CD NA, the following reagents, five microliters of T four DNA ligase buffer with 10 millimolar. A TP two microliters of DNTP Mix one microliter of T four DNA polymerase, one microliter of klino enzyme, and one microliter of T four PNK for end repair of the CD NA For the paired end library, add to 30 microliters of purified CD NA, these reagents 45 microliters of RNAs. D NS free water.
10 microliters of T four DNA ligase buffer with 10 millimolar. A TP four microliters of 10 millimolar. DNTP mix five microliters of T four DNA polymerase, one microliter of klino enzyme and five microliters of T four PNK incubate both tubes in the thermocycler at 20 degrees Celsius for 30 minutes after end repair.
The next step in this protocol is to add adenine bases to the three prime end of the DNA fragments for the single read library. Prepare the following reaction mixture 34 microliters of DNA sample five microliters of klino buffer, 10 microliters of DATP and one microliter of klino exo. For the paired end library, prepare the following reaction mixture 32 microliters of DNA sample five microliters of clin out buffer.
10 microliters of DATP and three microliters of clin out exo incubate both reaction mixtures at 37 degrees Celsius for 30 minutes prior to setting up the reactions for adapter ligation. Thaw the adapters on ice and dilute them one to 20 for the single read library. Use the Illumina chip sequence sample prep kit to prepare the following reaction mixture 10 microliters of DNA sample, one microliter of adapter oligo mix 15 microliters of two XDNA Ligase buffer and four microliters of DNA Ligase incubate for 15 minutes.
At room temperature for the paired end library, use the Illumina paired end sample prep kit to prepare the following reaction. Mix 10 microliters of DNA sample 10 microliters of PE adapter oligo mix 25 microliters of two XDNA ligase buffer and five microliters of DNA Ligase incubate for 15 minutes at 20 degrees Celsius at the end of the respective incubations. Clean up both ligation reactions using XMO columns.
Elute, the single read library and 44 microliters of water elute the paired end library with six microliters of illusion buffer, followed by another illusion in five microliters of eb. Perform a not one digestion only for the single read library. Incubate for two hours to overnight at 37 degrees Celsius after purifying the reaction using a ZY IMO column.
Elute the single read library with six microliters of buffer eb, followed by a second dilution with five microliters of eb. The adapter ligated, DNA for both libraries are now ready to be size selected. The size selection procedure for the adapter ligated DNA is performed using a 2%cyber safe EE from in Vitrogen to begin this procedure run program zero pre-run for two minutes.
After the pre-run load 10 microliters of the one KB plus ladder and the 2D NA samples. Fill the empty lanes with 10 microliters of water run program. One egel 2%run for 28 minutes.
When the run is complete size, select the 200 to 300 base pair of gel slices with a fresh razor blade. The number of genes identified by the various libraries was evaluated, and this table shows information on cluster numbers. Genes identified error rate and percent alignment of the single read and paired end libraries for both single read and paired end libraries.
The 10 nanogram T seven LA libraries identified almost the same number of genes as the 10 microgram UN amplified libraries with a TPM of 10 or more. In the case of the single read libraries, the 10 nanogram T seven LA library identified 100%of the 8, 500 genes identified by the 10 microgram UN amplified library for paradigm libraries. The 10 nanogram T seven LA library identified 86%of genes identified by the 10 microgram unpled library.
Libraries made from less than 10 nanograms were not able to identify as many genes. For example, in the single read protocol, the one nanogram T seven LA library identified only about 50%of the genes identified by the 10 microgram min amp library. Therefore, the lowest amount of total RNA for use with the T seven LA protocol should be limited to 10 nanograms mapping of a housekeeping gene.
GA DH shows that all T seven LA libraries made with at least 10 nanograms of starting RNA identified all exons, including the extreme five prime exon. The scale bar on the left for the single read libraries indicates 350 total reads. The scale bar in the center for the DEN libraries indicates 5, 000 total reads.
A comparison of the 10 nanogram T seven LA single read and paired end libraries with the min amp libraries shows a high degree of similarity. A spearman correlation of 0.90 was obtained between the single read 10 nanogram T seven LA library and the 10 microgram min amp library, and a correlation of 0.95 was obtained between the PEREN 10 nanogram T seven LA library and the 10 microgram min amp library. In addition, a comparison between the two single read and peren libraries made from 10 nanograms total RNA was made, and the very high correlation coefficient demonstrates that both types of libraries made using the T seven LA protocol produce a very similar gene expression signature.
Finally, since these libraries were prepared from human embryonic stem cell RNA 30 stem cells, specific genes were searched for among the libraries. Almost all of these genes were identified by the libraries made from at least 10 nanograms of starting total RNA, thus validating this protocol. While performing this protocol, it's important to remember to handle the small RNA samples with extreme caution to prevent degradation.
After watching this video, you should have a very good understanding of how to prepare Illumina, Mr.Aeq single read and Paradigm Libraries from very small amounts of starting material.
