A specialized cDNA library is made with
“U-select” primers, which home in on U-tags.
Theoretically possible, but because U-tag
library protocol is not a standard approach
for high-throughput sequencing, it may
need tweaking.
TRIBE
Catalytic domain of RNA-editing enzyme ADAR
from D. melanogaster
Converts adenosines to inosines at sites
targeted
by RNA-binding proteins
Standard RNA extraction
and preparation protocols
for RNA-seq
Possible in principle; TRIBE uses the same
standard deep-sequencing technique that has
been applied to single cells.
原文摘要：
TRIBE: Hijacking an RNA-Editing Enzyme to Identify Cell-Specific Targets of
RNA-Binding Proteins
RNA transcripts are bound and regulated by RNA-binding proteins (RBPs). Current
methods for identifying in vivo targets of an RBP are imperfect and not amenable
to examining small numbers of cells. To address these issues, we developed TRIBE
(targets of RNA-binding proteins identified by editing), a technique that
couples an RBP to the catalytic domain of the Drosophila RNA-editing enzyme ADAR
and expresses the fusion protein in vivo. RBP targets are marked with novel RNA
editing events and identified by sequencing RNA. We have used TRIBE to identify
the targets of three RBPs (Hrp48, dFMR1, and NonA). TRIBE compares favorably to
other methods, including CLIP, and we have identified RBP targets from as little
as 150 specific fly neurons. TRIBE can be performed without an antibody and in
small numbers of specific cells.
