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===rnase III=== Considerations to take into account for Rnase III fragmentation, and probably will produce dsRNA. *Invitrogen MEGAscript RNAi Kit is basically the same as MEGAscript T7 Kit **RNAi kit has more solutions/consumables, both contain same enzyme mix for T7 synthesis. Provides a method to create dsRNA which is highly similar to how the fragmentation library is being amplified, with two T7's on the ends of fragments. Either can be synthesized in sense and antisense direction in one reaction or in two different reactions. Below from MEGAscript RNAi kit manual. *[[File:Untitled.png|600px]] *Provides good template for RNase III which specifically cuts dsRNA. And shows this is already a used and well documented method in generation of siRNAs, but not for sequencing library amplification. *possible issues with dsRNA and higher Tm's? Espically since RNA-RNA and DNA-RNA structures have higher Tm's than DNA-DNA. paper showing some. [http://pubs.acs.org/doi/pdf/10.1021/bi00034a013 Relative Thermodynamic Stability of DNA, RNA, and DNA:RNA Hybrid Duplexes: Relationship with Base Composition and Structure] *RNase III fragmentation produces ends that are easy to ligate fragments to or use with polyA polymerase. End repair is not needed. *Only caveat is it seems to preferentially produce 25 ish bp fragments cutting from 5' and 3' ends. want to save 5' end since that is where the sequence tag is. Rnase III cuts from both 5' and 3' ends (for 21-25 bp fragments) *siRNA design for invitrogen kit places your sequence at the 5' end. *dicer is in the same family and makes same size nucleotide fragments *[http://www.nature.com/nature/journal/v475/n7355/abs/nature10198.html Dicer recognizes the 5′ end of RNA for efficient and accurate processing] *[http://products.invitrogen.com/ivgn/product/AM2290 invitrogen RNase III description] bc-RNase III *[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1935012/ Processing of Bacillus subtilis small cytoplasmic RNA: evidence for an additional endonuclease cleavage site] *[http://rnajournal.cshlp.org/content/6/8/1142.full.pdf Substrate recognition by a eukaryotic RNase III: the double-stranded RNA-binding domain of Rnt1p selectively binds RNA containing a 5'-AGNN-3' tetraloop.] *[http://www.ncbi.nlm.nih.gov/pubmed/9677377 Bacillus subtilis RNase III cleaves both 5'- and 3'-sites of the small cytoplasmic RNA precursor.] also showing random sites and 5' 3' sites *[http://www.pnas.org/content/74/3/984.full.pdf Nucleotide sequence surrounding a ribonuclease III processing site in bacteriophage T7 RNA] *RNase III only cuts dsRNA only dsRNA, not DNA-RNA: [http://www.jbc.org/content/249/4/1314.full.pdf Ribonuclease III Does Not Degrade Deoxyribonucleic Acid - Ribonucleic Acid Hybrids] NOT DNA-DNA, supercoiled DNA-DNA, ssRNA, doesn't remove phophates [http://www.neb.com/nebecomm/ManualFiles/manualE6146.pdf NEBNext® RNase III RNA Fragmentation Module] ssDNA?? VERY IMPORTANT!! can possibly block the 5' end by adding DNA primer prior to RNase III fragmentation to anneal and form DNA-RNA hybrid, since RNase III cannot degrade DNA-RNA, ssDNA or ssRNA strands. *This won't be an issue in downstream steps, including polyA addition, more specifically MMLV RT,and PCR. can add in 2x amount of forward primer during PCR since it will form primer dimer with first strand. MMLV can displace short DNA sequences (strand displacement) [http://www.neb.com/nebecomm/products/productM0253.asp can transcribe across RNA- DNA junction, stated in FAQs] searched: "mmlv strand displacement" [http://tools.invitrogen.com/content/sfs/productnotes/F_071215_SuperScript%20II-TS-TL-MKT-HL.pdf Invitrogen superscript II FAQ] "MMLV and SuperScript™ will strand displace to a small extent (<100 nt, about 30 nucleotides)"
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