GSM2418803: 0_1601; Homo sapiens; ChIP-Seq
Harvested cells were centrifuged at 300xg for 5 min, resuspended in PBS and centrifuged again.  The cell pellets were suspended in 20 ml PBS, made 1% HCHO (fresh ampule of 16% HCHO, methanol-free) and incubated 10 min at RT on a rotator.  Fixation was stopped by addition of 2.5 M glycine to a final concentration of 125 mM and incubated 5 min at RT on a rotator.  Fixed cells were centrifuged at 1000xg for 5 min and washed twice in PBS plus 50mM PMSF.  Cell pellets were suspended in 10 ml of ice-cold "swelling buffer" (10 mM KCl, 1 mM MgCl2, 0.1% NP-40, 1 mM DTT, 25 mM Hepes pH 7.8, made 0.5 mM PMSF and containing Roche COMPLETE), incubated 10 on ice, then centrifuged at 1000xg followed by plunging the cell pellets in liquid N2. The frozen cell pellets were resuspended in MNase (Micrococcal Nuclease) buffer (25 mM KCl, 4 mM MgCl2, 1 mM CaCl2, 50 mM Tris/HCl pH 7.4) and 10 U MNase per 1x106 cells were added. After 15 min, incubation at 37°C MNase was stopped by adding 10x Covaris buffer (100 mM Tris pH 8.0, 2 M NaCl, 10 mM EDTA, 5% N-lauroylsarcosine, 1% Na-deoxycholate, supplemented with protease inhibitors). The samples were sonicated for 15 min with the following parameters with a Covaris S2 system: burst 200, cycle 20%, and intensity 8. Following centrifugation the supernatant was collected and directly used for IP. After IgG preclearance the sheared chromatin was incubated overnight with protein G magnetic beads (Cell signaling, 9006) and 4 μg of anti-pan H3 (abcam, ab1791), H3K4me1 (abcam, ab8895), H3K9ac (active motif, 39917), H3K9me3 (abcam, ab8898), H3K27ac (abcam, ab4729), H3K36me3 (abcam, ab9050). After washes with 1x Covaris buffer (10 mM Tris–HCl, pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5% N-lauroylsarcosine, 0.1% Na–deoxycholate), high-salt-buffer (50 mM HEPES pH 7.9, 500 mM NaCl, 1mM EDTA, 1% Triton X-100, 0.1% Na–deoxycholate, 0.1% SDS), lithium buffer (20 mM Tris–HCl pH 8.0, 1 mM EDTA, 250 mM LiCl, 0.5% NP-40, 0.5% Na–deoxycholate) and 10 mM Tris–HCl, chromatin was eluted from the magnetic beads (elution buffer: 50 mM Tris pH 8.0, 1 mM EDTA, 1% SDS, 50 mM NaHCO3) and the crosslink was reversed overnight. After RNase A and proteinase K digestion, DNA was purified and cloned in a barcoded sequencing library for the Illumina sequencing platform. In brief, after DNA repair and A-addition NEBNext adapters (NEB, E7335) were ligated and digested with the USER enzyme. Barcodes (NEB, E7335) were introduced via PCR with a maximum of 14 cycles by the NEBNext polymerase (NEB, M0541). Size selection for mononucleosomal insert fragments was done with Ampure XP beads (Agencourt, A63880). Each ChIP-seq library was sequenced on the Illumina HiSeq 2000 with 50bp single-end, whereas the H3-chip samples were sequenced with 50 bp paired-ends.
GEO Accession
GSM2418803