GSM3402699: L428_H3K4me3; Homo sapiens; ChIP-Seq
For ChIP, 5x106 L428 cells were washed, resuspended at 3.3x106 cells/ml and initially crossklinked with 8.3 µl/ml DSG for 45 mins at room temperature. Cells were then washed 3 times with PBS using 500 g centrifugation, then subsequently crosslinked in 1% formaldehyde for 10 mins at room temperature. Quenching was carried out in by adding 1/10 volume of 2M glycine for a final concentration of 0.2M. Cells were Washed twice with ice-cold PBS, resuspended in1 ml 10 mM Hepes, 10 mM EDTA, 0.5 mM EGTA (all ph 8.0), 0.25 % Triton X100 and rotated 4 °C for 10 mins for lysis. Cells were then centrifuged at 500 g at 4 °C for 5 mins and nuclei resuspended in in 1ml 10 mM Hepes, 1mM EDTA, 0.5 mM EGTA (all pH 8.0), 200 mM NaCl, 0.01 % Triton X100 and rotated 4°C for 10 mins. Nuclei were then centrifuged at 500 g at 4°C for 5 mins and resuspended in 150 µl 25 mM Tris, 2 mM (both pH 8.0), 150 mM NaCl, 1 % Triton X100, 0.25 % SDS Sonication was performed using a Picoruptor sonicator (Diagenode, Belgium) using 30 30s on, 30s off cycles. Sonicated chromatin was centrifugated for 10 min at 16,000 g at 4 °C to pellet debris, then diluted in 300 µl 25 mM Tris, 2 mM (both pH 8.0), 150 mM NaCl, 1 % Triton X100, 0.25 %, 7.5 % Gylcerol ( final volume 450 µl ,0.083 % SDS, 5 % glycerol final concentration). All buffers contained 1:100 phosphatase inhibitor cocktail (Sigma-Aldrich, USA) and 0.1 mM PMSF. 5% of the sonicated chromatin was saved as input. 15 µl Protein G Dynabeads (Invitrogen, UK) were washed with 500 µl PBS + 0.0 2% Tween 20, resuspended in 15 µl PBS + 0.02 % Tween 20, 0.5 % BSA and 2 µg H3K4me3 millipore 04745 antibody, and rotated at 4 °C for 2 hours. Beads were washed with 500 µl PBS + 0.02 % Tween 20 and incubated with the sonicated chromatin (400 µl) on a rotating wheel overnight at 4 °C. Beads were then washed twice with 1ml 20 mM Tris, 2 mM EDTA 0.5 M (both pH 8.0), 150 mM NaCl, 1 % TritonX100, 0.1 % SDS, once with 1ml 20 mM Tris, 2 mM EDTA 0.5 M (both pH 8.0), 500 mM NaCl, 1 % TritonX100, 0.1 % SDS, once with 1 ml 10 mM Tris, 1 mM EDTA (both pH 8.0), 250 mM LiCl 0.5 % NP40, 0.5 % Na-deoxycholate and twice with 1ml 10 mM  Tris , 1 mM EDTA (both pH 8.0), 50 mM NaCl. Beads were subsequently incubated twice with 50 µl 100 mM NaHCO3, 1% SDS at 65 °C, and eluates reverse crosslinked overnight with 200 mM NaCl, 0.25 µg/µl proteinase K at 65 °C. Eluates were then incubated 1 hr with 0.1 µg/µl RNAse A at 37 °C. Phenol-chloroform extraction was performed by adding twice 100 µl equilibrated phenol-chloroform isoamyl alcohol (25:24:1), vortexing for 30s and spinning down at 16,000 g at 4°C for 5 mins, then adding 2.5 volumes 100% ethanol, 1/10 volume 5M NaCl and 1 µl glycogen, resuspending in 100 µl 0.1 H2O. Libraries were generated using the Kapa Hyperkit protocol (Kapa Biosystems, USA) according to manufacturer's instructions, using 16 cycles of amplification. 200-400 bp fragments were size-selected using a 2% agarose gel then subsequently purified using the QiaQuick Gel Extraction kit (QiaGen, Germany) according to manufacturer's instructions. High-throughput sequencing was performed on an Illumina HiSeq 2500 sequencer (Illumina, USA).
GEO Accession
GSM3402699