GSM8329693: human bone, input; Homo sapiens; ChIP-Seq
To study the changes of histone acetylome (H3K27ac) in human bone samples, chromatin was isolated from bones obtained from a surgery of hip replacement performed by Dr Liebregall at Hadassah hospital in Jerusalem. Briefly, 300 mgs of bones were thoroughly washed (X3) with cold PBS mixed with 1X protease inhibitor (cOmplete Mini, 11836153001), to clean samples from blood. Bones were crushed using a steel magnet bead in 1 ml of 10% Fetal bovine serum (FBS) using a TissueLyser LT instrument (Qiagen) set at 50 oscillations for 6 min (X2). After thorough disruption, chromatin released from bones was cross-linked by adding 37% formaldehyde (Sigma, F8775) to a final concentration of 1% and incubating in a rotator mixer for 15 min at room temperature. The reaction was stopped by adding 2.5 M glycine to a final concentration of 0.125 M and incubating in a rotator mixer for additional 5 min at room temperature. The fixed samples were washed 2 times using cold PBS mixed with 1X protease inhibitor by centrifuging at 4°C in a speed of 500 rcf for 5 min. To lyse cells, pellets were resuspended in 1 ml of SDS lysis buffer (1% SDS, 10 mM EDTA of pH 8, 50 mM Tris HCl of pH 8.1) and incubated on ice for 25 min. The fragmentation of chromatin DNA was achieved by sonicating the samples for 20 cycles with 30 sec pulse on high and 30 sec pulse off using a water-bath sonicator to the length of 200–600 bp. The sheared chromatin was then centrifuged at 4°C in a speed of 20000 rcf for 15 min. After running electrophoresis on agarose gel to check sonication results, the supernatant containing sheared chromatin was diluted 1:10 in a dilution buffer (1.1% Triton X-100, 1.2 mM EDTA of pH 8, 16.7 mM Tris-HCL of pH 8.1, 167 mM NaCl) containing 1X protease inhibitor. To set up ChIP reactions, the diluted chromatin was mixed with 5 µg of anti-H3K27ac (CMA309, Kimura) and 10 ul of the Magna-ChIP protein A magnetic beads (16-661, Millipore), and the mix was incubated for 14 hours on a rotator mixer at 4°C. Beads were then sequentially washed with rotation for 2 min once with Low Salt Immune Complex Wash Buffer (1% Triton X-100, 2 mM EDTA of pH 8, 20 mM Tris-HCL of pH 8.1, 150 mM NaCl, 0.1% SDS, 1X protease inhibitor), High Salt Immune Complex Wash Buffer (1% Triton X-100, 2 mM EDTA of pH 8, 20 mM Tris-HCL of pH 8.1, 500 mM NaCl, 0.1% SDS, 1X protease inhibitor), LiCl Immune Complex Wash Buffer (1 mM EDTA of pH 8, 10 mM Tris-HCL of pH 8.1, 1% NP-40, 250 mM LiCl, 1% Deoxycholate, 1X protease inhibitor), and once without rotation with TE Buffer (1 mM EDTA of pH 8, 10 mM Tris-HCL of pH 8.1, 1X protease inhibitor). DNA was then eluted from the beads by incubation in 300 μL elution buffer (1 mM EDTA of pH 8, 10 mM Tris-HCL of pH 8.1, 200 mM NaCl, 1% SDS) for 5 min at room temperature. Then, the samples were incubated with 1 μL RNase A (10109142001, Merck) for 30 min at 37°C with 600 rpm shaking. The histone-DNA crosslinks were reversed by the addition of 2 µl of 20 mg/ml Proteinase K (25530049, Thermo Fisher Scientific) and incubation at 65°C for overnight. Finally, DNA was purified using a phenol-chloroform protocol. Briefly, one volume of the lower phase of phenol:chloroform:isoamyl alcohol was added and the samples were vortexed thoroughly for approximately 25 sec. The samples were centrifuged for 10 minutes at 15000 rcf. The upper phase was transferred to a new tube, mixed with 3 M sodium acetate (1:10), 2 µl of 20 mg/ml glycogen and two volumes of 100% cold ethanol. The samples were incubated at -20°C for overnight to precipitate the DNA from the samples. The samples were centrifuged at 4°C in a speed of 15000 rcf for 30 min. The pellets were washed with 70% ethanol and centrifuged at 4°C in a speed of 15000 rcf for 15 min. The pellets were dried in the hood for 15 minutes and resuspended with 10 mM Tris pH 8. The concentration of DNA was quantified using HS Qubit. ChIP-seq libraries were prepared as previously described in high-throughput ChIP protocol (Blecher-Gonen et. al., 2013) with minor modifications. Briefly, 10 ng of DNA were purified again using SPRI beads and amplified by 14 PCR cycles following end repairing, A-base addition and adapter ligation. The quality of libraries was checked on TapeStation (Agilent) and Qubit (Invitrogen). All ChIP-seq libraries were sequenced in one lane of Illumina® NextSeq® 500 system.