Daniel:Notebook/RNAFISH/2014-7-11

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Revision as of 21:08, 11 July 2014 by >Djacobse (→‎Results)
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Probe Design

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Probe List

Rui sent me a list of genes to design probes for. Here it is:

  • GRIK4 Glutamate Receptor, IK-4
  • GPC6 HS glypican -coreceptor for growth factors
  • ERBB4 ERBB4
  • GRIK1 Glutamate receptor, IK-1
  • DNER Notch-like grow factor receptor
  • DCC netrin 1 receptor
  • POU6F2 POU TF
  • FOXP2 TF
  • GAD1 glutamate decarboxylase 1
  • GAD2 glutamate decarboxylase 2
  • CA10 carbonic anhydrase

Workflow

  1. Identify genomic coordinates using UCSC genome browser, hg38
  2. Download the CDS Exon sequence using the tables tab of the genome browser
  3. Run MATLAB script that outputs only the sequence can also trim the sequence to 8kbp (Stellaris maximum)
    1. MATLAB script
  4. Paste sequence into Stellaris website
    1. Currently using 25 probes per gene
  5. Copy/paste sequences into a .txt file

Results

The following link contains a list of the probes generated by Stellaris. There are 25 20mers per gene. All probes are taken from the first 8000bp unless the gene contained fewer than 8000bp, in which case the whole gene is used. 8000 is the limit imposed by Stellaris on their website. Also below is the probe sequences in a standalone file. The gene order is the same as the probe list, and a blank line separates genes. The oligo list is closer to the format IDT needs for ordering, although all probes need to have a /5AmMC6 / to add an amino modifier to the C6 carbon on the 5' end (for ARES labeling).