Characterization of RNA - Animal Models of Acute Neurological Injuries: Injury and Mechanistic Assessments - page 142

Experiment Type and
Sample Selection

Variations in the construction of RNA-seq libraries can generate
profi les from different RNA populations within a given sample.
Size fractionation of total RNA can separate miRNA, Piwi-
interacting RNA (piRNA), and other small regulatory species from
the majority of mRNAs and rRNAs. This can be performed by
polyacrylamide gel electrophoresis (PAGE). As many of the mature
forms of these RNAs have a size range of ~19-32 nt, they may be
sequenced in their entirety with most of the short read high-
throughput sequencing technologies. Conversely, mRNA sequenc-
ing requires isolation of larger polyadenylated mRNA species,
usually using an oligo-dT-based capture method prior to perform-
ing the sequencing protocol. While none of the reads will cover the
entirety of the mRNA, due to the large number of sequencing
fragments we can generate long contigs from signifi cant overlaps
between each read. It is important to note that those RNAs (i.e.,
many noncoding RNAs) lacking a polyA tail are excluded from
sequencing experiments constructed in this manner. The primary
reason for both of these selection procedures is to reduce the con-
tent of ribosomal RNA in the pool of RNAs to be sequenced,
which if not removed, would make up the bulk of all sequenced
fragments—a limitation of the technology as it stands. Fortunately,
each sequencing platform is experiencing rapid innovation, and the
fi delity and read length of the sequences is improving.

(a) Template preparation:
The fi rst step in deep sequencing is the preparation of repre-
sentative templates (a library) for each sample. The two primary
methods involve either clonally amplifi ed fragments (454,
Illumina, SOLiD3) or direct sequencing of RNA (Helicos).
Isolated molecules are captured and immobilized on a support
surface such that they are at low enough density to remain
distinct from their neighbors. The sequence of bases can be
decoded by stepwise imaging of fl uorescent bases. The sequencing
and imaging methods are fundamentally platform specifi c and
not covered here as they are not usually amenable to experi-
menter manipulation or variation.
(b) Alignment and quantitation:
For RNAseq experiments, the sequenced fragments are normally
aligned to a reference genome of interest. Your choice of refer-
ence will largely determine the level of detail that is recovered
from the sequencing data. The most common option is to use
a well-annotated genomic reference genome (e.g., mm9 for
mouse and rn4 for rat); however, references constructed from
transcriptome databases may also be used to identify known
and novel RNA species. The tools for performing the alignment
are usually provided along with the sequencing technology
used; however, there has been considerable advancement in the
commercial and open-source sectors to build aligners that rival
and/or surpass those offered by the sequencing technology

3.2.2. Procedures