GSM9041492: F14_tsc_HU_g1_3_nodox_H3K27me3_chip; Mus musculus; ChIP-Seq
ChIP 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. ChIP DNA was then reverse crosslinked in 0.5% SDS and 100mM NaCl overnight at 65°C, followed by a 1-h RNaseA (3L; Thermo Scientific EN0531) treatment at 37°C and a 2.5-h Proteinase K (10L; Invitrogen 25530015) treatment at 56°C. DNA was then extracted with 1 volume of phenol:chloroform:isoamyl alcohol (Sigma-Aldrich P3803) and precipitated with 2 volumes 100% ethanol, 1/10 volume 3M sodium-acetate pH 5.4, and 1/1000 volume linear acrylamide (Invitrogen AM9520) overnight at -20°C. Precipitated DNA was then extracted with a 30-minute max speed spin at 4°C, washed once with ice-cold 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, 150-bp sequencing was performed on an Illumina NextSeq1000 platform with a 100-cycle NextSeq 1000/2000 P2 Reagents v3 kit (Illumina 20046811)