illumina cBot, Системное руководство

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cBot 2 System Guide
25
Denature Libraries and Add Optional PhiX Control
The library loading concentration depends on the libraries to be sequenced. The following
instructions apply to supported Illumina libraries and assume an insert size typical for the
associated library type. Make sure that you dilute to a concentration appropriate for the
library type.
} A DNA loading concentration that is too high causes reduced %PF.
}
A DNA loading concentration that is too low causes reduced %PF and a high
percentage of duplicates that negatively affect depth of coverage.
1 Dilute the library or pooled libraries to the appropriate concentration.
Library Type Dilution
TruSeq DNA PCR-Free Dilute to 1–2 nM in RSB.
TruSeq Nano DNA Dilute to 2–3 nM in RSB.
Nextera Rapid Capture Exome
TruSeq Stranded Total RNA
TruSeq Stranded mRNA
2 [Optional] Spike-in 1% nondenatured Illumina PhiX control to nondenatured libraries.
Library Type Spike-In
TruSeq DNA PCR-Free Add 5 µl 100–200 pM PhiX to 45 µl 1–2 nM library.
TruSeq Nano DNA Add 5 µl 200–300 pM PhiX to 45 µl 2–3 nM library.
Nextera Rapid Capture Exome
TruSeq Stranded Total RNA
TruSeq Stranded mRNA
3 Number the tubes of an 8-tube strip:
} For clustering with sample tracking—From the key end, label the tubes #8 through
#1.
} For clustering without sample tracking—Label the tubes #1 through #8.
4 Denature the library in the 8-tube strip as follows:
a Add 5 µl nondenatured library into the bottom of each well.
b Add 5 µl freshly diluted 0.1 N NaOH. Slowly pipette to mix.
c Incubate at room temperature for 8 minutes.
d Add 5 µl 200 mM Tris-HCl pH 8.0. Slowly pipette to mix.
5 Set aside on ice until you are ready to add the ExAmp master mix.
CAUTION
Prepare and add the ExAmp master mix within 30 minutes .
Prepare the cBot Reagent Plate
1 Invert to mix.
2 Vortex to dislodge trapped air bubbles.
3 Tap on a hard surface to collect reagent droplets. Alternatively, pulse centrifuge.
4 Set aside on ice.