Editing BioMicroCenter:RNA HTL (section)

== Digital Gene Expression (DGE) ==
=== 3'Digital Gene Expression ===
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  |+ '''HT-3'DGE'''
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  ! style="text-align: center; width: 150px;" | Parameter
  ! style="text-align: center; width: 300px;" | General requirements
  ! style="text-align: center; width: 300px;" | [[BioMicroCenter:RNA_HTL#RNA_Pre-load_Submissions|Pre-load requirements]]
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  | style="text-align: center; height: 2em;" | SAMPLE INPUT
  | colspan="2" style="text-align: center; height: 2em;" | Intact RNA ([[BioMicroCenter:RIN|RIN]] &ge; 7)
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  | style="text-align: center; height: 2em;" | RANGE OF INPUT
  | style="text-align: center; height: 2em;" | 5ng - 15ng
  | style="text-align: center; height: 2em;" | [[BioMicroCenter:RNA_HTL#Normalization|Normalized]] samples<br>(2ng/&micro;L)
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  | style="text-align: center; height: 2em;" | SUBMISSION VOLUME
  | style="text-align: center; height: 2em;" | >10 &micro;L nuclease free water or 10mM Tris pH8 (EB)
  | style="text-align: center; height: 2em;" | Exactly 5 &micro;L nuclease free water
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  | style="text-align: center; height: 2em;" | UNIT
  | colspan="2" style="text-align: center; height: 2em;" | 24 samples<br>96 samples
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  | style="text-align: center; height: 2em;" | PLATE SETUP
  | colspan="2" style="text-align: center; height: 2em;" | Samples should be arrayed by row in a 96-well full-skirt plate (Axygen)
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  | style="text-align: center; height: 2em;" | SEQUENCING RECOMMENDATIONS
  | colspan="2" style="text-align: center; height: 2em;" | NextSeq
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  | style="text-align: center; height: 2em;" | INDEX AVAILABILITY
  | colspan="2" style="text-align: center; height: 2em;" |16 [[BioMicroCenter:RNA_HTL#Unique_versus_Combinatorial_Dual_Indexing|Unique Dual Indexes]] <br> 96 [[BioMicroCenter:RNA_HTL#Unique_versus_Combinatorial_Dual_Indexing|Combinatorial Dual Indexes]]
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  | style="text-align: center; height: 2em;" | INCLUDED
  | colspan="2" style="text-align: center; height: 2em;" |cDNA generation of poly(A) RNA<br>QC check of cDNA<br>Library preparation (Nextera XT)<br>QC check of final libraries
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  | style="text-align: center; height: 2em;" | ADDITIONAL SERVICES AVAILABLE
  | style="text-align: center; height: 2em;" | Sample QC <BR> Sample cleaning <BR> Sample arraying
  | style="text-align: center; height: 2em;" | [[BioMicroCenter:RNA_HTL#RNA_Pre-load_Submissions|N/A]]
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  | style="text-align: center; height: 2em;" | SUBMISSION FORM
  | colspan="2" style="text-align: center; height: 2em;" | MIT - [https://mit.ilabsolutions.com/service_item/new/3381?spt_id=4110 ilabs] <BR> External - [[BioMicroCenter:Forms|form]]
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  | style="text-align: center; height: 2em;" | PRICING
  | colspan="2" style="text-align: center; height: 2em;" | [[BioMicroCenter:Pricing#ILLUMINA_LIBRARY_-_DNA|LINK]]
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<br>
:High-Throughput 3' Digital Gene Expression (HT3DGE) uses a combination of molecular tagged indexes, SMARTseq chemistry and Nextera Tagmentation to produce libraries derived from the 3' ends of transcripts.<br><br>
:The protocol is based on [http://dx.doi.org/10.1101/003236/ Soumillon et al.,] as part of a collaboration with the KI High-Throughput Screening Core. In limiting the sequence space used by the samples, fewer reads should be required for a good transcriptome.<BR><BR>

:HT3DGE uses a very early indexing step to tag each sample with a "well ID" and a molecular ID. Once tagged the samples are immediately pooled to minimize costs but failed samples cannot be easily reprepped and are not identifiable until sequencing. As such, the method is best suited for experiments where the NUMBER of samples is not limiting (material can be). The 3' nature of the protocol also limits the utility of this method in splicing applications but it should be more robust to using imperfect RNA ([[BioMicroCenter:RIN|RIN]] 7+ instead of 9+ for standard RNAseq) <BR><BR>

:Analysis of HT3DGE is not a standard method supported by most open source platforms. We have built pipelines to work with this data, but working closely with the Bioinformatics team is highly recommended.
 |<br>[[image:DGE_workflow.jpg|thumb|right|275px|Standard 3'DGE workflow.]]
[[image:HT3DGE_gene_example.jpg|thumb|right|400px|Example 3'DGE Data]]<BR><BR>
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