9. Analyze the qPCR results. Use the Cq values measured from
the qPCR assay to estimate the library concentration for each
sample, using the C q values measured from the DNA standards
to calibrate. To convert from nM to ng/μL, we estimate an
average fragment base pair length of 220 bp.
3.4 Assess Library
Quality
1. Dilute 2–3 μL of the library (1:4) in 10 mM Tris, pH 8, or
0.1Â TE, pH 8.
2. Run 1 μL in a DNA High Sensitivity Chip.
3. Check that the electropherogram shows a narrow distribution
with a peak size approximately 300 bp.
4. Interpretation: If a peak at ~80 bp (primers) or 128 bp
(adaptor-dimer) is shown in the bioanalyzer traces, then bring
up the sample volume (Subheading 3.2, step 47) to 50 μL
exactly with nuclease-free water and repeat the AMPure XP
Bead cleanup step (Subheading 3.2, steps 39–47).
3.5 Pooling
Multiplexed Sample
Libraries
The number of samples you pool together is dependent on the
sequencing platform and experimental parameters. Once to the
pooling step, you must further decide what concentration to pool
your samples. Pooling concentration varies by sequencing center
used; if using your own bench top machine, the Illumina site has a
pooling calculator that will best determine the concentration based
on platform and other experimental conditions. You should dilute
the concentration of the pooled library to match the needs of the
external sequencing service (if using), or, if you are performing the
sequencing yourself, to one of the following concentrations: 4 nM,
2 nM, 1 nM, or 0.5 nM, depending on amount of available starting
material.
3.6 Prepare Libraries
for Sequencing
The following protocol is adapted from the NextSeq System Denature and Dilute Libraries Guide (Document #15048776v09). This
step is only intended for users who are planning to perform the
sequencing run on the Illumina NextSeq instrument directly.
Those who plan to submit pooled libraries to external sequencing
services should skip to Subheading 3.8.
The library volume and loading concentration differ depending
on the version of NCS you are running (see Note 16): For NCS v
1.2 or earlier, use a Library volume of 3 mL at a concentration of
3 pM. For NCS v 1.3 or later, use a Library volume of 1.3 mL at a
concentration of 1.8 pM. The protocol that we describe here
assumes NCS v 1.3 or later (see Note 17):
1. Prepare a Fresh Dilution of NaOH.
Combine the following volumes in a microcentrifuge tube.
The result is 1 mL of 0.2 M NaOH:
RNAseq Workflow
497
the qPCR assay to estimate the library concentration for each
sample, using the C q values measured from the DNA standards
to calibrate. To convert from nM to ng/μL, we estimate an
average fragment base pair length of 220 bp.
3.4 Assess Library
Quality
1. Dilute 2–3 μL of the library (1:4) in 10 mM Tris, pH 8, or
0.1Â TE, pH 8.
2. Run 1 μL in a DNA High Sensitivity Chip.
3. Check that the electropherogram shows a narrow distribution
with a peak size approximately 300 bp.
4. Interpretation: If a peak at ~80 bp (primers) or 128 bp
(adaptor-dimer) is shown in the bioanalyzer traces, then bring
up the sample volume (Subheading 3.2, step 47) to 50 μL
exactly with nuclease-free water and repeat the AMPure XP
Bead cleanup step (Subheading 3.2, steps 39–47).
3.5 Pooling
Multiplexed Sample
Libraries
The number of samples you pool together is dependent on the
sequencing platform and experimental parameters. Once to the
pooling step, you must further decide what concentration to pool
your samples. Pooling concentration varies by sequencing center
used; if using your own bench top machine, the Illumina site has a
pooling calculator that will best determine the concentration based
on platform and other experimental conditions. You should dilute
the concentration of the pooled library to match the needs of the
external sequencing service (if using), or, if you are performing the
sequencing yourself, to one of the following concentrations: 4 nM,
2 nM, 1 nM, or 0.5 nM, depending on amount of available starting
material.
3.6 Prepare Libraries
for Sequencing
The following protocol is adapted from the NextSeq System Denature and Dilute Libraries Guide (Document #15048776v09). This
step is only intended for users who are planning to perform the
sequencing run on the Illumina NextSeq instrument directly.
Those who plan to submit pooled libraries to external sequencing
services should skip to Subheading 3.8.
The library volume and loading concentration differ depending
on the version of NCS you are running (see Note 16): For NCS v
1.2 or earlier, use a Library volume of 3 mL at a concentration of
3 pM. For NCS v 1.3 or later, use a Library volume of 1.3 mL at a
concentration of 1.8 pM. The protocol that we describe here
assumes NCS v 1.3 or later (see Note 17):
1. Prepare a Fresh Dilution of NaOH.
Combine the following volumes in a microcentrifuge tube.
The result is 1 mL of 0.2 M NaOH:
RNAseq Workflow
497