title: GSM8332741: K562-2; Homo sapiens; OTHER
library_construction_protocol: 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 10 minutes. The end-repair step was not performed. Libraries were then generated with the NEBNext Ultra™ II DNA Library Kit (NEB, E7805S) without purification. After ligated with adaptors, DNA fragments are selected with 20 μLs of Streptavidin T1 beads (Invitrogen™, 65602) by incubation at room temperature for 1h with agitation. The beads were washed 3 times each with: Low Salt wash buffer [0.1% SDS (Invitrogen™, AM9820), 0.1% Triton X-100 (Sigma-Aldrich, T8787-50ML), 2 mM EDTA pH 8.0 (Invitrogen™, AM9260G), 20 mM Tris-HCl pH 8.0 (Invitrogen™, 15568025), 150 mM NaCl (Thermo Fisher Scientific, AM9759)], 1x B&W buffer (5 mM Tris-HCl pH 7.5 (Invitrogen™, 15567027), 0.5 mM EDTA pH 8.0 (Invitrogen™, AM9260G), 1M NaCl (Thermo Fisher Scientific, AM9759)), and 1x PBS pH 7.4 (Gibco™, 70011044) with 0.1% Triton X-100 (Sigma-Aldrich, T8787-50ML). 50 μLs of the pre-mixed reagents (NEB, E7805S) including 25 μLs of 2x PCR Master Mix, 5 μLs of Universal Primer, and 5 μLs of Primer Mix-Index 1 from the NEBNext Ultra II Single Indexing Kit (NEB, E7335S) were added to the beads. The amplification was conducted with an initial denaturation of 95°C for 2 minutes then cycled 15 times with a 98°C -denaturation step for 10 seconds and a 68°C combined annealing and extension step for 90 seconds. PCR products were purified with 0.75x AMPure XP Beads (Beckman, A63880), eluted in 20 μLs of UltraPure™ DNase/RNase-Free distilled water (Thermo Scientific, 10977023), and quantified with the Qubit dsDNA HS Assay Kit (Invitrogen™, Q32851). Each library was paired-end sequenced for 150 cycles on each end on an Illumina NovaSeq 6000.