sequencing runs when generating libraries with fewer than
96 samples per sequencing run, it is also helpful to keep a lab
document of what primers have been used.
13. The number of PCR cycles should be adjusted based on RNA
input. If 20 ng of mRNA is the starting input from Subheading
3.2, it is recommended to perform 14 cycles of PCR.
14. It is important to limit the number of PCR cycles to avoid
overamplification. If overamplification occurs, larger molecular
weight products (>500 bp) will appear on the bioanalyzer
trace.
15. We include at least one reaction for the 1:32000 and 1:64000
dilutions as input for the KAPA Quantification reaction to
ensure that the DNA concentration falls within the range of
one of the standards provided in the kit. When following this
protocol, we have used the outlined dilution range and dilution
buffer as it improves accuracy and ensures that the diluted
samples will fall within the range of the standards provided.
Ideally, all reactions should be set up in triplicate to assess
technical reproducibility.
16. Typically, it is important that not more than 1 mM NaOH is in
the final solution after diluting with HT1. However, introducing 200 mM Tris–HCl ensures that NaOH is fully hydrolyzed
in the final solution. As a result, template hybridization is not
affected even if the final NaOH concentration after diluting
with HT1 is greater than 1 mM.
17. WARNING: The reagents in the sequencing kit contain
potentially hazardous chemicals. Personal injury can occur
through inhalation, ingestion, skin contact, and eye contact.
Wear protective equipment, including eye protection, gloves,
and laboratory coat appropriate for risk of exposure. Handle
used reagents as chemical waste and discard in accordance with
applicable regional, national, and local laws and regulations.
For additional environmental, health, and safety information,
see the SDS at support.illumina.com/sds.html.
18. In place of RSB, you can use 10 mM Tris–HCl, pH 8.5 with
0.1% Tween 20.
19. A low-concentration PhiX control library spike-in is used at
$1% as a sequencing control.
20. The following protocol is adapted from the Illumina NextSeq
500 System Guide (Document #15046563v06). 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.
RNAseq Workflow
509
96 samples per sequencing run, it is also helpful to keep a lab
document of what primers have been used.
13. The number of PCR cycles should be adjusted based on RNA
input. If 20 ng of mRNA is the starting input from Subheading
3.2, it is recommended to perform 14 cycles of PCR.
14. It is important to limit the number of PCR cycles to avoid
overamplification. If overamplification occurs, larger molecular
weight products (>500 bp) will appear on the bioanalyzer
trace.
15. We include at least one reaction for the 1:32000 and 1:64000
dilutions as input for the KAPA Quantification reaction to
ensure that the DNA concentration falls within the range of
one of the standards provided in the kit. When following this
protocol, we have used the outlined dilution range and dilution
buffer as it improves accuracy and ensures that the diluted
samples will fall within the range of the standards provided.
Ideally, all reactions should be set up in triplicate to assess
technical reproducibility.
16. Typically, it is important that not more than 1 mM NaOH is in
the final solution after diluting with HT1. However, introducing 200 mM Tris–HCl ensures that NaOH is fully hydrolyzed
in the final solution. As a result, template hybridization is not
affected even if the final NaOH concentration after diluting
with HT1 is greater than 1 mM.
17. WARNING: The reagents in the sequencing kit contain
potentially hazardous chemicals. Personal injury can occur
through inhalation, ingestion, skin contact, and eye contact.
Wear protective equipment, including eye protection, gloves,
and laboratory coat appropriate for risk of exposure. Handle
used reagents as chemical waste and discard in accordance with
applicable regional, national, and local laws and regulations.
For additional environmental, health, and safety information,
see the SDS at support.illumina.com/sds.html.
18. In place of RSB, you can use 10 mM Tris–HCl, pH 8.5 with
0.1% Tween 20.
19. A low-concentration PhiX control library spike-in is used at
$1% as a sequencing control.
20. The following protocol is adapted from the Illumina NextSeq
500 System Guide (Document #15046563v06). 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.
RNAseq Workflow
509