5. This is the adapter that gets ligated to 5
0 ends of target RNA
molecules. Most of the oligo is composed of deoxyribonucleotides to improve stability, while five ribonucleotides are
included at the 3
0 end to improve ligation efficiency. Store at
À80
C.
6. Betaine makes the reaction mix cloudy; this is not a cause for
concern.
7. The addition of betaine, the long denaturation steps, and the
setting of the reaction volume as 100 μL are all steps taken to
ensure that GC-rich sequences are fully represented in the final
libraries. See Ref. 6 for further explanation.
8. Primers SSS401 and SSS402 are short primers that anneal to
the outer portions of the ~60 nucleotide Illumina adapter
sequences. This PCR enriches for molecules that contain fulllength adapters to ensure optimal attachment to the Illumina
flow-cell for sequencing.
9. The ratio of bead volume to sample volume is important
because this determines the size selection properties [7, 8]. A
ratio of 1.8 is appropriate for purifying away primers from PCR
products. If sample volumes vary significantly from those indicated in this protocol, the volume of beads should be adjusted
accordingly to maintain the correct ratio.
10. When preparing libraries, use the minimum number of PCR
cycles that produce sufficient finished library for sequencing.
This reduces the extent to which PCR bias and jackpotting
produce skewed abundances in your libraries. However, for
the purposes of testing the library size with a diagnostic PCR,
the number of cycles can be higher (25 in this case) because the
products will not be sequenced.
11. Add ethidium bromide to both the gel and the running buffer
to ensure that small fragments can be visualized. Library reactions should give smears or bands that run higher than the
primer-dimer present in the no-template control reactions.
Library fragment size may vary depending on fragmentation
method used.
12. Significant care must be taken to prevent cross-contamination
by trace amounts of Illumina library amplicon on laboratory
equipment, since all Illumina sequencing libraries contain
adapter sequences that are identical in their outer portions.
All gel boxes and trays should be thoroughly cleaned before
use in preparative steps such as Subheading 3.7 and after all
steps that involve amplified library molecules (Subheadings 3.6
and 3.7). For best practice, designate different areas of the lab
for preamplification and postamplification procedures and
maintain separate pipets, racks, and other equipment for each
530
M. Carla Martini et al.
0 ends of target RNA
molecules. Most of the oligo is composed of deoxyribonucleotides to improve stability, while five ribonucleotides are
included at the 3
0 end to improve ligation efficiency. Store at
À80
C.
6. Betaine makes the reaction mix cloudy; this is not a cause for
concern.
7. The addition of betaine, the long denaturation steps, and the
setting of the reaction volume as 100 μL are all steps taken to
ensure that GC-rich sequences are fully represented in the final
libraries. See Ref. 6 for further explanation.
8. Primers SSS401 and SSS402 are short primers that anneal to
the outer portions of the ~60 nucleotide Illumina adapter
sequences. This PCR enriches for molecules that contain fulllength adapters to ensure optimal attachment to the Illumina
flow-cell for sequencing.
9. The ratio of bead volume to sample volume is important
because this determines the size selection properties [7, 8]. A
ratio of 1.8 is appropriate for purifying away primers from PCR
products. If sample volumes vary significantly from those indicated in this protocol, the volume of beads should be adjusted
accordingly to maintain the correct ratio.
10. When preparing libraries, use the minimum number of PCR
cycles that produce sufficient finished library for sequencing.
This reduces the extent to which PCR bias and jackpotting
produce skewed abundances in your libraries. However, for
the purposes of testing the library size with a diagnostic PCR,
the number of cycles can be higher (25 in this case) because the
products will not be sequenced.
11. Add ethidium bromide to both the gel and the running buffer
to ensure that small fragments can be visualized. Library reactions should give smears or bands that run higher than the
primer-dimer present in the no-template control reactions.
Library fragment size may vary depending on fragmentation
method used.
12. Significant care must be taken to prevent cross-contamination
by trace amounts of Illumina library amplicon on laboratory
equipment, since all Illumina sequencing libraries contain
adapter sequences that are identical in their outer portions.
All gel boxes and trays should be thoroughly cleaned before
use in preparative steps such as Subheading 3.7 and after all
steps that involve amplified library molecules (Subheadings 3.6
and 3.7). For best practice, designate different areas of the lab
for preamplification and postamplification procedures and
maintain separate pipets, racks, and other equipment for each
530
M. Carla Martini et al.