procedure requires considerably more affinity-purified complexes than the amount needed for the RNA-SEQ library
construction [31].
10. TRAP yield from tissues expressing FLAG-RPL18 protein in
nearly all cell types is about 500 ng of RNA per mL of ground
tissue. TRAP RNA yield ranges between 10 and 150 ng per mL
of tissue when the FLAG-RPL18 protein is expressed in a
limited number of cells (e.g.: root atrichoblast, cortical cells
of the root meristematic zone, root vasculature); therefore, a
significantly higher amount of tissue should be processed in
order to obtain ~500 ng of RNA [46].
11. The quality of the RNA starting material is crucial for the
success of RNA-SEQ library preparation. The RNA integrity
number (RIN) provides robust and reliable prediction of RNA
integrity. The RIN is calculated by a method that automatically
selects features from signal measurements recorded with an
Agilent 2100 Bioanalyzer and constructs regression models
based on a Bayesian learning technique. RIN values range
from 10 (intact RNA) to 1 (totally degraded RNA). In order
to proceed with RNA-SEQ library preparation, RIN should be
8 or higher and the ratio of the 25S signal to the 18S signal
should be about 1.8.
12. Take into consideration that for low initial RNA amount it is
recommended to dilute adapters (e.g., starting with 400 ng of
total RNA, we used a 1:8 dilution of adapters in TE buffer with
good results) to avoid an excess of adapters that can lead to the
formation of contaminating adapter dimers or oligomers (see
Fig. 3c).
13. Commercial beads can be collected and diluted to 3Â the
original volume in 20% PEG/2.5 M NaCl without affecting
the purification.
14. TRAP RNA-SEQ libraries of sufficient quality for Illumina
RNA-SEQ should produce a signal distribution between
200 and 500 bp with a maximum at approximately 260 bp,
such as those illustrated in Fig. 3c. Prior to running DNA
samples in the Bioanalyzer, they can be evaluated in an ethidium bromide-stained 1.5% (w/v) agarose gel when working
with abundant RNA, or an acrylamide gel stained with SYBR
Gold. A distribution of DNA fragments between 200 and
500 bp with no adapter dimers should be observed (Fig. 3c).
15. The HiSeq3000/4000 system can yield approximately
300–400 million single-end reads (101 bp length) allowing
the sequencing of up to 32 multiplexed libraries in a
single lane.
TRAP-SEQ Analysis of Long Noncoding RNAs
469
construction [31].
10. TRAP yield from tissues expressing FLAG-RPL18 protein in
nearly all cell types is about 500 ng of RNA per mL of ground
tissue. TRAP RNA yield ranges between 10 and 150 ng per mL
of tissue when the FLAG-RPL18 protein is expressed in a
limited number of cells (e.g.: root atrichoblast, cortical cells
of the root meristematic zone, root vasculature); therefore, a
significantly higher amount of tissue should be processed in
order to obtain ~500 ng of RNA [46].
11. The quality of the RNA starting material is crucial for the
success of RNA-SEQ library preparation. The RNA integrity
number (RIN) provides robust and reliable prediction of RNA
integrity. The RIN is calculated by a method that automatically
selects features from signal measurements recorded with an
Agilent 2100 Bioanalyzer and constructs regression models
based on a Bayesian learning technique. RIN values range
from 10 (intact RNA) to 1 (totally degraded RNA). In order
to proceed with RNA-SEQ library preparation, RIN should be
8 or higher and the ratio of the 25S signal to the 18S signal
should be about 1.8.
12. Take into consideration that for low initial RNA amount it is
recommended to dilute adapters (e.g., starting with 400 ng of
total RNA, we used a 1:8 dilution of adapters in TE buffer with
good results) to avoid an excess of adapters that can lead to the
formation of contaminating adapter dimers or oligomers (see
Fig. 3c).
13. Commercial beads can be collected and diluted to 3Â the
original volume in 20% PEG/2.5 M NaCl without affecting
the purification.
14. TRAP RNA-SEQ libraries of sufficient quality for Illumina
RNA-SEQ should produce a signal distribution between
200 and 500 bp with a maximum at approximately 260 bp,
such as those illustrated in Fig. 3c. Prior to running DNA
samples in the Bioanalyzer, they can be evaluated in an ethidium bromide-stained 1.5% (w/v) agarose gel when working
with abundant RNA, or an acrylamide gel stained with SYBR
Gold. A distribution of DNA fragments between 200 and
500 bp with no adapter dimers should be observed (Fig. 3c).
15. The HiSeq3000/4000 system can yield approximately
300–400 million single-end reads (101 bp length) allowing
the sequencing of up to 32 multiplexed libraries in a
single lane.
TRAP-SEQ Analysis of Long Noncoding RNAs
469