Fig. 3 Experimental scheme and yield evaluation for translating ribosome affinity purification (TRAP). (a) TRAP
takes advantage of cells expressing an epitope-tagged ribosomal protein (FLAG-tagged RPL18 in plants). After
homogenization, polysomes are stabilized in polysome extraction buffer. mRNAs and lncRNAs associated with
ribosomes are captured to anti-FLAG (α-FLAG) magnetic beads. The beads are collected using a magnet and
washed extensively before RNA extraction for directional RNA-sequencing library preparation. Optionally,
ribosomal complexes can be eluted by competition with an excess of 3XFLAG peptide. (b) Western blot of rice
extracts obtained from seedlings that express p35S:FLAG-RPL18 or wild-type control in different steps of
TRAP, using α-FLAG-coupled IgG Dynabeads or α-FLAG agarose magnetic beads for polysome purification.
The blot was developed with α-FLAG antibodies. α-FLAG coupled to IgG Dynabeads yields more FLAG-RPL18
compared to α-FLAG in agarose beads. (c) mRNA libraries obtained after TRAP from rice roots expressing
p35S:FLAG-RPL18 or an INTACT construct as a control [47]. A final amplification step of 17 cycles was used to
test the presence of mRNA not specifically bound to α-FLAG IgG Dynabeads. Obtained products, without final
cleanup, were separated in an 8% (w/v) acrylamide gel and stained with SYBR Gold. Library fragments and
adapters are indicated
TRAP-SEQ Analysis of Long Noncoding RNAs
455
takes advantage of cells expressing an epitope-tagged ribosomal protein (FLAG-tagged RPL18 in plants). After
homogenization, polysomes are stabilized in polysome extraction buffer. mRNAs and lncRNAs associated with
ribosomes are captured to anti-FLAG (α-FLAG) magnetic beads. The beads are collected using a magnet and
washed extensively before RNA extraction for directional RNA-sequencing library preparation. Optionally,
ribosomal complexes can be eluted by competition with an excess of 3XFLAG peptide. (b) Western blot of rice
extracts obtained from seedlings that express p35S:FLAG-RPL18 or wild-type control in different steps of
TRAP, using α-FLAG-coupled IgG Dynabeads or α-FLAG agarose magnetic beads for polysome purification.
The blot was developed with α-FLAG antibodies. α-FLAG coupled to IgG Dynabeads yields more FLAG-RPL18
compared to α-FLAG in agarose beads. (c) mRNA libraries obtained after TRAP from rice roots expressing
p35S:FLAG-RPL18 or an INTACT construct as a control [47]. A final amplification step of 17 cycles was used to
test the presence of mRNA not specifically bound to α-FLAG IgG Dynabeads. Obtained products, without final
cleanup, were separated in an 8% (w/v) acrylamide gel and stained with SYBR Gold. Library fragments and
adapters are indicated
TRAP-SEQ Analysis of Long Noncoding RNAs
455