title: GSM6805229: C/B TSC EZH2 biol rep 2; Mus musculus; ChIP-Seq
library_construction_protocol: For all ChIP DNA elution steps, washed beads were resuspended in Elution buffer (50mM Tris-HCl pH 8.0, 10mM EDTA, 1% SDS) and placed on a 65°C heat block for 17 minutes with frequent vortexing. RAD21 ChIP DNA was eluted for 1 hour under the same conditions. ChIP DNA was then reverse crosslinked in 0.5% SDS and 100mM NaCl overnight at 65°C, followed by a 1-hour RNaseA (3uL; Thermo Scientific, cat #: EN0531) treatment at 37°C and a 2.5-hour Proteinase K (10uL; Invitrogen, cat #: 25530015) treatment at 56°C. DNA was then extracted with 1 volume of phenol:chloroform:isoamyl alcohol (Sigma-Aldrich, cat #: P3803) and precipitated with 2 volumes 100% ethanol, 1/10 volume 3M sodium-acetate pH 5.4, and 1/1000 volume linear acrylamide (Invitrogen, cat #: AM9520) overnight at -20°C. Precipitated DNA was then extracted with a 30-minute max speed spin at 4°C, washed once with 80% ethanol, and resuspended in TE. ChIP-Seq libraries were prepared with NEBNext End Repair Module (NEB, cat #: E6050S), A-tailing by Klenow Fragment (3'--5' exo-; NEB, cat #: M0212S), and TruSeq 6-bp index adaptor ligation by Quick ligase (NEB, cat #: M2200S), and NEBNext High-Fidelity 2X PCR Master Mix (NEB, cat #: M0541S). All DNA clean-up steps were performed with AMPure XP beads (Beckman Coulter, cat #: A63880). Single-end, 75-bp sequencing was performed using an Illumina NextSeq 500/550 High Output v2.5 kit (Illumina, cat #: 20024906) on a NextSeq 500 System.