12. Place the 20 pM library on ice until you are ready to proceed to
final dilution.
13. Dilute the denatured 20 pM library solution to 1.8 pM as
follows:
(a) Denatured library solution (117 μL).
(b) Prechilled HT1 (1183 μL).
The total volume is 1.3 mL at 1.8 pM.
14. Invert to mix library and then pulse centrifuge.
15. Thaw a tube of 10 nM PhiX library (see Note 19) stock
(10 μL/tube).
16. Combine the following volumes in a microcentrifuge tube:
(a) 10 nM PhiX (10 μL).
(b) RSB (15 μL).
The total volume is 25 μL at 4 nM.
17. Vortex briefly and then pulse centrifuge. The 4 nM PhiX can be
stored at À25
C to À15
C for up to 3 months.
18. Combine the following volumes in a microcentrifuge tube:
(a) 4 nM PhiX (5 μL).
(b) 0.2 N NaOH, freshly diluted (5 μL).
19. Vortex briefly and then pulse centrifuge.
20. Incubate at room temperature for 5 min.
21. Add 5 μL of 200 mM Tris–HCl, pH 7.0.
22. Vortex briefly and then centrifuge at 280 Â g for 1 min.
23. Add 985 μL of prechilled HT1 to the tube of denatured PhiX.
The total volume is 1 mL at 20 pM.
24. Dilute the denatured 20 pM PhiX to 1.8 pM as follows. The
total volume is 1.3 mL at 1.8 pM:
(a) Denatured PhiX (117 μL).
(b) Prechilled HT1 (1183 μL).
25. Invert to mix and then centrifuge at 280 Â g for 1 min. The
denatured 1.8 pM PhiX can be stored at À25
C to À15
C for
up to 2 weeks. After 2 weeks, cluster numbers tend to decrease.
26. Combine the following volumes of denatured PhiX control
and denatured library (using 1.5 pM PhiX for High Output
Kits):
(a) Denatured and diluted PhiX control at 1.5 pM (13 μL).
(b) Denatured and diluted library (1287 μL).
27. Set aside on ice until you are ready to load it onto the reagent
cartridge. The library and PhiX mixture provide a PhiX spike-in
of 0.5–2.0%. Actual PhiX percentage varies depending upon
the quality and quantity of the library pool.
RNAseq Workflow
499
final dilution.
13. Dilute the denatured 20 pM library solution to 1.8 pM as
follows:
(a) Denatured library solution (117 μL).
(b) Prechilled HT1 (1183 μL).
The total volume is 1.3 mL at 1.8 pM.
14. Invert to mix library and then pulse centrifuge.
15. Thaw a tube of 10 nM PhiX library (see Note 19) stock
(10 μL/tube).
16. Combine the following volumes in a microcentrifuge tube:
(a) 10 nM PhiX (10 μL).
(b) RSB (15 μL).
The total volume is 25 μL at 4 nM.
17. Vortex briefly and then pulse centrifuge. The 4 nM PhiX can be
stored at À25
C to À15
C for up to 3 months.
18. Combine the following volumes in a microcentrifuge tube:
(a) 4 nM PhiX (5 μL).
(b) 0.2 N NaOH, freshly diluted (5 μL).
19. Vortex briefly and then pulse centrifuge.
20. Incubate at room temperature for 5 min.
21. Add 5 μL of 200 mM Tris–HCl, pH 7.0.
22. Vortex briefly and then centrifuge at 280 Â g for 1 min.
23. Add 985 μL of prechilled HT1 to the tube of denatured PhiX.
The total volume is 1 mL at 20 pM.
24. Dilute the denatured 20 pM PhiX to 1.8 pM as follows. The
total volume is 1.3 mL at 1.8 pM:
(a) Denatured PhiX (117 μL).
(b) Prechilled HT1 (1183 μL).
25. Invert to mix and then centrifuge at 280 Â g for 1 min. The
denatured 1.8 pM PhiX can be stored at À25
C to À15
C for
up to 2 weeks. After 2 weeks, cluster numbers tend to decrease.
26. Combine the following volumes of denatured PhiX control
and denatured library (using 1.5 pM PhiX for High Output
Kits):
(a) Denatured and diluted PhiX control at 1.5 pM (13 μL).
(b) Denatured and diluted library (1287 μL).
27. Set aside on ice until you are ready to load it onto the reagent
cartridge. The library and PhiX mixture provide a PhiX spike-in
of 0.5–2.0%. Actual PhiX percentage varies depending upon
the quality and quantity of the library pool.
RNAseq Workflow
499