Category:Methods and Protocols: Difference between revisions

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Rough overview of HeliScope CAGE

We started HeliScope CAGE with manual operation (with multi(8)-channel pipet), and are achieving automated preparation of the CAGE libraries to scale up the number of profiles (96 RNAs can be treated at once). The sequencing results of the libraries are computationally post-processed in the following steps:

• Discard apparent artifacts or too-short sequences by filterSMS (a utility of Helicos software, HeliSphere)
• Discard the CAGE tags derived from the ribosomal DNA repeating unit, which is not contained in the genome assembly, by rRNAdust (developed by Timo Lassmann)
• Align the remained CAGE tags with the genome sequences with DELVE (developed by Timo Lassmann), which generate BAM files containing a single mapped position per read with mapping quality and alignments.
• Aggregate the 5'-end of mapped CAGE tags as CAGE transcription starting site (CTSS)
• Loaded them into the genome browsers (ZENBU F5 instance or the local mirror of UCSC Genome Browser), as well as inclusion into file release.

Individual protocols

• RNA extraction

There's various RNA extraction protocols used for FANTOM5 samples...

• • OP-RNA-extraction-totalRNA-TRIzol-ethanol-v1.0
  • OP-RNA-extraction-totalRNA-TRIzol-isopropanol-v1.0
  • OP-RNA-extraction-totalRNA-miRNeasy_Mini-v1.0
  • OP-RNA-extraction-totalRNA-RiboPure_minelute_cleanup-v1.0
  • OP-RNA-extraction-totalRNA-ToTALLY-RNA-v1.0

• HeliScopeCAGE library construction
  • manual library preparation (standard) OP-HELISCOPE-CAGE-v3.12
  • manual library preparation (low quantity) OP-HELISCOPE-CAGE-v5.0
  • automated library preparation OP-HELISCOPE-CAGE-v4.0
• HeliScope sequencing OP-HELISCOPE-sequencing-v1.0
• Filtering with HeliSphere/filterSMS OP-HELICOS-CAGE-Filtering-v1.0
• CAGEscan library construction
  • CAGEscan libraries are prepared using the nanoCAGE protocol, version 1.4 (OP-SOLEXA-nanoCAGE-Direct-v1.4), and sequenced paired-end according to standard procedures on Illumina Genome Analyzer IIx.
• rRNA dust on ribosomal DNA repeating unit
• alignment of the CAGE reads with the genome
• 5'-end based aggregation of CAGE tags (termed level1 in FANTOM4 and CTSS in FANTOM3)