GSM9235524: Monocytes, solvent 40 minutes, ChIP replicate 1; Homo sapiens; ChIP-Seq
ChIP-seq : After treatment of 20,000,000 THP-1 cells, nuclear proteins were cross-linked to genomic DNA by adding formaldehyde directly to the medium to a final concentration of 1% and incubating at room temperature for 10 min on a rocking platform. Cross-linking was stopped by adding glycine to a final concentration of 0.125 M and incubating at room temperature for 10 min on a rocking platform. The cells were collected by centrifugation and washed twice with ice cold PBS. The cell pellets were subsequently resuspended twice in 10 ml cell lysis buffer (0.1% SDS, 1 mM EDTA, 150 mM NaCl, 1% Triton X-100, 0.1% sodium deoxycholate, protease inhibitors, 50 mM HEPES-KOH, pH 7.5) and once in 10 ml nuclear lysis buffer (1% SDS, 1 mM EDTA, 150 mM NaCl, 1% Triton X-100, 0.1% sodium deoxycholate, protease inhibitors, 50 mM HEPES-KOH, pH 7.5). After two washes with cell lysis buffer, the chromatin pellet was resuspended in 700 µl of SDS lysis buffer (1% SDS, 10 mM EDTA, protease inhibitors, 50 mM Tris-HCl, pH 8.1) and the lysates were sonicated in a Bioruptor Plus (Diagenode) to result in DNA fragments of 200 to 500 bp. Cellular debris was removed by centrifugation. 340 µl aliquots of the lysate were diluted 1:5 in IP dilution buffer (1% Triton X-100, 2 mM EDTA, 150 mM NaCl, protease inhibitors, 20 mM Tris-HCl, pH 7.5). 25 µl anti-VDR antibody (Cell Signaling Technology, #12550) was coated to 60 µl Dynabeads Protein G (Invitrogen) in an overnight incubation at 4 °C. The pre-formed bead-antibody complexes were then washed twice with beads wash buffer (0.1% Triton X-100, PBS, protease inhibitors) and added to the chromatin aliquots. The samples were incubated for overnight at 4 °C on a rotating wheel to form and collect the immuno-complexes. The beads were washed sequentially for 5 min on a rotating wheel with 1 ml of the following buffers, each: twice cell lysis buffer, once high salt buffer (0.1% SDS, 1% Triton X-100, 1 mM EDTA, 350 mM NaCl, 0.1% sodium deoxycholate, 50 mM HEPES-KOH, pH 7.5), once ChIP wash buffer (250 mM LiCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 1 mM EDTA, 10 mM Tris-HCl, pH 8.0) and twice 1 ml TE buffer (1 mM EDTA, 10 mM Tris-HCl, pH 8.0). Then, the immune complexes were eluted using 250 µl ChIP elution buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl, pH 7.5) at 37 °C for 30 min with rotation. The elution was repeated with a 10 min rotation and the supernatants were combined. Immune complexes were reverse cross-linked at 50-65 °C for 2-4 h in the presence of proteinase K (Fermentas) in a final concentration of 40 µg/ml. Genomic DNA was isolated with the ChIP DNA Clean&Concentrator Kit (Zymo Research) ChIP-seq  libraries were sequenced on an Illumina HiSeq 2000 (Illumina) at 50 bp read length using standard protocols at the Gene Core facility of the EMBL (Heidelberg, Germany)