Editing BioMicroCenter:RNAseq (section)

== RNAseq Methods ==

{| border=1
 ! Amount of RNA
 ! Quality of RNA
 ! Method Recommended
 |-
 | >100ng || RIN:9.0 || Illumina TruSeq
 |-
 | >1ug || RIN:5.0 || Epicenter RiboZero
 |- 
 | 10pg-100ng || RIN:9.0 || Clontech SMARTer Low Input
 |- 
 | 1ng-1ug || RIN:5.0 || NuGEN Ovation RNA-Seq System V2
|}

===[http://www.illumina.com/products/truseq_rna_sample_prep_kit_v2.ilmn Illumina TruSeq]===
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Illumina's TruSeq chemistry is the primary RNAseq method used in the BioMicro Center. This chemistry uses polyT beads to isolate the mRNA from the rRNA and tRNA. The use of these beads requires that the RNA be of very high quality or only the 3' end of transcripts will be isolated. Purified mRNA is then fragmented with metal and random priming is used to convert the sample to cDNA. Once double-stranded cDNA is generated, the sample is transferred to the [[BioMicroCenter:SPRI-Works|SPRIworks]] for the remainder of sample preparation.
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[[Image:BMC_RNAseqMethod.png|thumb|right|200px|TruSeq Chemistry]]
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[[Image:BMC_IlluminaRNAseq.png|thumb|right|300px|Sample Data]]
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=== [http://www.epibio.com/item.asp?ID=589 EpiCenter RiboZero] ===
For samples with degraded RNA or samples where you are interested in looking at non-polyA RNAs, the BioMicro Center utilizes the Epicenter ribominus kit. This kit uses *check me* magnetic beads coupled to rRNA and tRNA sequences to remove these sequences from the solution. The remaining mRNA fragments can then be converted in to cDNA. Once double-stranded cDNA is generated, the sample is transferred to the [[BioMicroCenter:SPRI-Works|SPRIworks]] for the remainder of sample preparation.

=== [http://www.clontech.com/US/Products/cDNA_Synthesis_and_Library_Construction/cDNA_Synthesis_Kits/Ultra_Low_Input_RNA_cDNA_Synthesis Clontech SMARTer Low-Input] ===
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For samples with less then 100ng of input, the BioMicro Center utilizes the [http://www.clontech.com/US/Products/cDNA_Synthesis_and_Library_Construction/cDNA_Synthesis_Kits/Ultra_Low_Input_RNA_cDNA_Synthesis Clontech SMARTer system]. This system differs from the TruSeq chemisry in that it begins with cDNA generation using polyT priming and proprietary chemistry. The use of polyT priming requires the RNA to be of high quality. Full length double-stranded cDNAs are generated and amplified by PCR. These cDNAs are then fragmented and transferred to the [[BioMicroCenter:SPRI-Works|SPRIworks]] for the remainder of sample preparation. Data from this system is of similar quality to samples created with Illumina TruSeq chemistry.
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[[Image:BMC_ClontechChemistry.png|thumb|right|200px|Clontech system.<BR>Image from Clonetech]]
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=== [http://www.nugeninc.com/nugen/index.cfm/products/cs/ngs/rna-seq-v2/ NuGEN Ovation RNAseq System V2] === 
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For samples with less then 100ng of input and restricted input amounts, our kit of choice is the NuGEN Ovation. This kit utilizes non-random nonamers designed to not amplify ribosomal RNA to create double stranded cDNA fragments. The fragmented cDNA is then transferred to the [[BioMicroCenter:SPRI-Works|SPRIworks]] for the remainder of sample preparation. The downside of this kit is that the non-random nonamers cannot amplify all areas of the genome and certain portions of genes are often lost. Still, for many samples, this kit is the only way to generate RNAseq libraries.
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[[Image:BMC_SPIA.gif|thumb|right|200px|NuGEN SPIA Chemistry. <BR> Image from NuGen.]]
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