Brandon:LabNotes/Project1/2013-7-10
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Testing new method of RNA processing, No RNase III, No polyA polymerase (PAP)
- In performing the Tn5 accessibility method, the most troubling steps are when RNase III is used for fragmentation and polyA polymerase is used to add polyA tails to the 3' end, so the T20VN_PE_R primer can be used to add adaptors/barcodes etc. Thus this protocol avoids the usage of both enzymes. RNase III does not seem to have a good shelf life and does not function correctly if it is not taken care of, and stops working approximately 3 months after purchase. PAP is a extremely robust enzyme, and adds polyA to most segments. Most of the unmapped reads are polyA tailed fragments with the sequencing reads consisting of polyA sequences.
- Changes:
- Another robust method of generating cDNA will be used, using random hexamers for priming and MMLV for RT
- RNA in the RNA/cDNA hybrid will be degraged with RNase H, and heated for denaturing. Next, extension with BST 2.0 or Klenow exo- will be done with a DNA primer to the 5' end.
- DNA will will need to be fragmented, but this time a custom transpsome will be created that only has the nextera B ends on it, the 3' end of fragments were barcodes are attached.
Sequences information
Protocol
- check is have reagents etc
1. generating custom nextera transposome
2.