A disadvantage of the previously described TRAP protocol [46]
is the large amount of starting material of transgenic animal or plant
tissue required to obtain acceptable yields of RNA material. The
former procedure made use of antibodies coupled to agarose beads
and an elution step; for example, for a FLAG-tagged ribosomal
protein, TRAP assays are performed with anti-FLAG-agarose beads
and elution is achieved by competition with an excess of 3X FLAG
peptide (Fig. 3b). Then, RNA is purified from the eluted fraction to
obtain the TRAP RNA. Here, we describe an optimized procedure
that makes use of magnetic beads and eliminates the elution step,
which significantly increases RNA yield as compared to the previously reported TRAP procedure. We also provide details of library
construction and bioinformatics analysis of the RNA-seq data followed to identify ribosome-associated lncRNAs (Fig. 3c) [47].
2 Materials
2.1 Biological
Material, Reagents,
and Kits
1. Biological material: Fresh tissue expressing a tagged ribosomal
protein (e.g., CaMV35S:FLAG-RPL18 Medicago truncatula
roots). The amount of tissue depends on the number of cells
expressing the fusion protein.
2. Dynabeads™ Protein G for immunoprecipitation (Invitrogen).
3. Monoclonal anti-FLAG antibody (see Note 1).
4. TRIzol Reagent (Invitrogen).
5. Miracloth.
6. Glycogen (molecular biology grade).
7. TruSeq
® Stranded mRNA Library Prep (Illumina, cat. #
20020610) or YourSeq Dual (FT + 3
0 -DGE) RNAseq Library
Kits (Amaryllis Nucleics, https://amaryllisnucleics.com/kits/
dual-rnaseq-library-kit) or a homemade version as
described [48].
8. Agencourt AMPure XP Beads (Beckman Coulter).
9. Agilent RNA 6000 Pico kit.
10. Agilent DNA 1000 kit.
2.2 Stock Buffers
and Solutions
The following solutions need to be autoclaved and can be stored at
room temperature for a month. Unless otherwise indicated, the
solvent for solutions is molecular biology-grade water.
1. 2 M Tris–HCl, pH 9.0. Adjust the pH using concentrated HCl
(see Note 2).
2. 2 M KCl.
3. 0.5 M Ethylene glycol-bis (2-aminoethylether)-N,N,N
0 ,N0 -tetraacetic acid (EGTA), pH 8.0. Adjust the pH using
NaOH pellets (see Note 3).
TRAP-SEQ Analysis of Long Noncoding RNAs
457
is the large amount of starting material of transgenic animal or plant
tissue required to obtain acceptable yields of RNA material. The
former procedure made use of antibodies coupled to agarose beads
and an elution step; for example, for a FLAG-tagged ribosomal
protein, TRAP assays are performed with anti-FLAG-agarose beads
and elution is achieved by competition with an excess of 3X FLAG
peptide (Fig. 3b). Then, RNA is purified from the eluted fraction to
obtain the TRAP RNA. Here, we describe an optimized procedure
that makes use of magnetic beads and eliminates the elution step,
which significantly increases RNA yield as compared to the previously reported TRAP procedure. We also provide details of library
construction and bioinformatics analysis of the RNA-seq data followed to identify ribosome-associated lncRNAs (Fig. 3c) [47].
2 Materials
2.1 Biological
Material, Reagents,
and Kits
1. Biological material: Fresh tissue expressing a tagged ribosomal
protein (e.g., CaMV35S:FLAG-RPL18 Medicago truncatula
roots). The amount of tissue depends on the number of cells
expressing the fusion protein.
2. Dynabeads™ Protein G for immunoprecipitation (Invitrogen).
3. Monoclonal anti-FLAG antibody (see Note 1).
4. TRIzol Reagent (Invitrogen).
5. Miracloth.
6. Glycogen (molecular biology grade).
7. TruSeq
® Stranded mRNA Library Prep (Illumina, cat. #
20020610) or YourSeq Dual (FT + 3
0 -DGE) RNAseq Library
Kits (Amaryllis Nucleics, https://amaryllisnucleics.com/kits/
dual-rnaseq-library-kit) or a homemade version as
described [48].
8. Agencourt AMPure XP Beads (Beckman Coulter).
9. Agilent RNA 6000 Pico kit.
10. Agilent DNA 1000 kit.
2.2 Stock Buffers
and Solutions
The following solutions need to be autoclaved and can be stored at
room temperature for a month. Unless otherwise indicated, the
solvent for solutions is molecular biology-grade water.
1. 2 M Tris–HCl, pH 9.0. Adjust the pH using concentrated HCl
(see Note 2).
2. 2 M KCl.
3. 0.5 M Ethylene glycol-bis (2-aminoethylether)-N,N,N
0 ,N0 -tetraacetic acid (EGTA), pH 8.0. Adjust the pH using
NaOH pellets (see Note 3).
TRAP-SEQ Analysis of Long Noncoding RNAs
457