Tagmented libraries
were barcoded and amplified using KAPA HiFI polymerase and a collection
of Nextera XT 12mer dual unique index sequencing primers (purchased
from IDT and supplied by CZ-Biohub). First, 1.2 μL of a master
mix containing 0.08 μL of KAPA HiFi (1 U/μL), 0.8 μL
of 5× buffer (manufacturer supplied), 0.12 μL of 10 mM
dNTP mix (2.5 mM each), and 0.2 μL of H2O was added
to each 2 μL SDS-halted Tn5 reaction using the Mantis liquid
handler. Next, 0.8 μL of unique i5/i7 primer mix (2.5 μM
each) was transferred from source plates to sample using the Mosquito
instrument. Reaction plates were sealed, briefly vortexed, collected
by centrifugation, and amplified with the following thermal cycler
conditions: 72 °C, 3 min; 95 °C, 30 s; 12 × [98 °C,
10 s; 55 °C, 15 s; 72 °C, 1 min]; 72 °C, 5 min. Resulting
libraries were pooled (with the Mosquito) and treated with AMPure
XP magnetic beads at a ratio of 0.8:1 beads:sample volume to purify
DNA (Beckman Coulter, Brea, CA, USA). Library yield and quality were
determined by TapeStation electrophoresis with an HSD1000 ScreenTapes
(Agilent Technologies).
were barcoded and amplified using KAPA HiFI polymerase and a collection
of Nextera XT 12mer dual unique index sequencing primers (purchased
from IDT and supplied by CZ-Biohub). First, 1.2 μL of a master
mix containing 0.08 μL of KAPA HiFi (1 U/μL), 0.8 μL
of 5× buffer (manufacturer supplied), 0.12 μL of 10 mM
dNTP mix (2.5 mM each), and 0.2 μL of H2O was added
to each 2 μL SDS-halted Tn5 reaction using the Mantis liquid
handler. Next, 0.8 μL of unique i5/i7 primer mix (2.5 μM
each) was transferred from source plates to sample using the Mosquito
instrument. Reaction plates were sealed, briefly vortexed, collected
by centrifugation, and amplified with the following thermal cycler
conditions: 72 °C, 3 min; 95 °C, 30 s; 12 × [98 °C,
10 s; 55 °C, 15 s; 72 °C, 1 min]; 72 °C, 5 min. Resulting
libraries were pooled (with the Mosquito) and treated with AMPure
XP magnetic beads at a ratio of 0.8:1 beads:sample volume to purify
DNA (Beckman Coulter, Brea, CA, USA). Library yield and quality were
determined by TapeStation electrophoresis with an HSD1000 ScreenTapes
(Agilent Technologies).