How to load the Nextseq 550 sequencing machine once you have the sequencing pool ready

• Unfreeze the Pool, PhiX, Flowcell, HT1 and Reagent Cartridge (Illumina)
• HT1 and Reagent Cartridge can thawed in water to save some time.
• Take 2 empty 1,5ml Tubes and dilute NaOH(dilute to 0.2N) and Tris (dilute to 200 nM) with UltraPure Water (total 100µl dilution).
• Take 2 empty 1,5 ml Tubes for Denaturation and final Dilution.
• Denaturation: mix 5µl the ~ 4nM Pool and 5 µl freshly diluted 0.2N NaOH
• Vortex briefly and then centrifuge at 280x g for 1 minute
• Incubate at room temperature for 5 minutes
• Add 5 µl Tris-HCl, pH 7
• Vortex briefly and then centrifuge at 280x g for 1 minute
• Dilute denatured Pool to 20 pM with prechilled HT1. How much HT1 you need depends on the pool conc. (see in Pooling sheet)
• Vortex briefly and then centrifuge at 280x g for 1 minute
• Place the 20 pM pools on ice until you are ready to proceed to final dilution