GSM6997608: PBMC, Input, C9ORF72; Homo sapiens; ChIP-Seq
The samples were centrifuged at 13,800 x g for 10 min at 4 °C and the supernatant was mixed with chromatin buffer (provided) in a 1:1 ratio. Sheared chromatin was pre-cleared with 1 % BSA blocked protein A and G mag Sepharose Xtra magnetic beads (GE Healthcare) and subsequently incubated overnight at 4 °C on a rotator with antibody-labelled beads (10 µg antibody, H3K27m3 Merck or Chrom Pure Rabbit IgG from Jackson Immuno Research). Beads were washed with buffers of increasing stringency (low salt (0.1 % SDS, 1 % Triton X-100, 2 mM EDTA, 20 mM Tris-HCl [pH 8], 150 mM NaCl) to high salt (0.1 % SDS, 1 % Triton X-100, 2 mM EDTA, 500 mM NaCl)) and bound chromatin complexes were eluted (100 mM NaHCO3, 1% SDS). The DNA-antibody bead complex was reverse-crosslinked at 65 °C for 5 h (1300 rpm) and digested with proteinase K for further 2 h at 65 °C (1300 rpm) to remove proteins. Finally, DNA was purified using the ChIP DNA clean and concentrator kit (Zymo research). Libraries were generated using the NEBNext Ultra II DNA Library Prep Kit (Illumina) and subjected to single-end sequencing (101 bp) on a HiSeq2500 platform (Illumina).