The DNA was released from the beads with the NEBNext®
Ultra™ II FS DNA Module (NEB, E7810S) using twice the
reaction volume and a fragmentation time of 5 minutes. The end
repair step was not performed. Libraries were then generated with
the NxSeq® UltraLow DNA Library Kit (Lucigen, 15012-1) up to
the final AMPure XP Bead purification before amplification. Each
sample was eluted in 50 uLs Nuclease-free water, and added to 10 uLs
of Dynabeads™ MyOne™ Streptavidin T1 beads suspended
in 50 uLs 1x PBS pH 7.4 with 0.1% Triton X-100. The selection was
performed at room temperature for 1 hour. Beads were washed 2 times
with 500 uLs Low Salt buffer [0.1% SDS (Invitrogen, AM9820), 0.1%
Triton™ X-100, 2 mM EDTA, 20 mM Tris-HCl buffer, pH 8
(Invitrogen, 15568025), 150 mM NaCl], 2 times with 500 uLs 1x
B&W buffer (5 mM Tris-HCl pH 7.5, 0.5 mM EDTA, 1M NaCl), and 2
times with 500 uLs 1x PBS pH 7.4 with 0.1% Triton™ X-100.
Library amplification was then performed with the NxSeq®
UltraLow DNA Library Kit as directed.
Each library was paired end sequenced for 100 cycles on each
end on an lllumina HiSeq 4000 or NovaSeq 6000.
Ultra™ II FS DNA Module (NEB, E7810S) using twice the
reaction volume and a fragmentation time of 5 minutes. The end
repair step was not performed. Libraries were then generated with
the NxSeq® UltraLow DNA Library Kit (Lucigen, 15012-1) up to
the final AMPure XP Bead purification before amplification. Each
sample was eluted in 50 uLs Nuclease-free water, and added to 10 uLs
of Dynabeads™ MyOne™ Streptavidin T1 beads suspended
in 50 uLs 1x PBS pH 7.4 with 0.1% Triton X-100. The selection was
performed at room temperature for 1 hour. Beads were washed 2 times
with 500 uLs Low Salt buffer [0.1% SDS (Invitrogen, AM9820), 0.1%
Triton™ X-100, 2 mM EDTA, 20 mM Tris-HCl buffer, pH 8
(Invitrogen, 15568025), 150 mM NaCl], 2 times with 500 uLs 1x
B&W buffer (5 mM Tris-HCl pH 7.5, 0.5 mM EDTA, 1M NaCl), and 2
times with 500 uLs 1x PBS pH 7.4 with 0.1% Triton™ X-100.
Library amplification was then performed with the NxSeq®
UltraLow DNA Library Kit as directed.
Each library was paired end sequenced for 100 cycles on each
end on an lllumina HiSeq 4000 or NovaSeq 6000.