Illumina HiSeq 2000 paired end sequencing; Investigating the requirement of H3K14 and H3P16 for optimal H3K4me3
Investigating the requirement of H3K14 and H3P16 for optimal H3K4me3
Yeast cells were grown at 30 degrees C, shaking at 200 rpm to exponential phase in YPD medium (1 % yeast extract (Difco), 1 % bactopeptone supplemented with 2 % glucose). Cells were grown overnight in 5 ml cultures before dilution to 0.2 OD600 (0.5 x 107 cells/ml) in the appropriate volume of fresh media. Cells were harvested during exponential growth at 0.5 OD600 (1.25 x 107 cells/ml) by centrifugation (3000 rpm, 5 min). Cells grown to OD600 0.5 in 50 ml of appropriate media were fixed with 1 % formaldehyde in 45 ml PBS for 30 min at 22 degrees C followed by addition of glycine to 125 mM for 5 min. Cell pellets were collected by centrifugation (3000 rpm, 5 min) before washing twice with 10 ml cold PBS. Cells were resuspended in 500 ul cold FA-150 buffer (10 mM HEPES pH 7.9, 150 mM NaCl, 0.1 % SDS, 0.1 % sodium deoxycholate, 1 % Triton X-100) and broken using 1 ml glass beads on a MagnaLyser (Roche) at 4 degrees C. Sample volume was increased to 2 ml with FA-150 buffer before shearing of the fixed chromatin by sonication using a biorupter (Diagenode, 30 min, 1 min on, 20s off, medium setting). Chromatin was cleared by centrifugation (10 000 rpm, 15 min, 4 degrees C) and incubated with antibody (H3 Millipore/07-690 1:40 dilution, H3K4me3 Millipore/05-745R 1:40 dilution) in 1.5 ml siliconised Eppendorf tubes for 15-20 h rotating at 4 degrees C. Bound chromatin was immunoprecipitated for 90 min at 22 degrees C with 50 ul protein A-Sepharose pre-blocked with sonicated salmon sperm DNA. Beads and attached chromatin were pelleted by centrifugation (2600 rpm, 1 min) and washed with TSE-150 buffer (20 mM Tris-Cl pH 8.0, 150 mM NaCl, 2 mM EDTA, 0.1 % SDS, 1 % Triton X-100) for 3 min, TSE-500 buffer (20 mM Tris-Cl pH 8.0, 500 mM NaCl, 2 mM EDTA, 0.1 % SDS, 1 % Triton X-100) for 3 min, LiCl buffer (0.25 M LiCl, 10 mM Tris-Cl pH 8.0, 1 mM EDTA, 1 % dioxycholate, 1 % NP-40) for 15 min and twice with TE, all at 22 degrees C. After washing, chromatin was eluted from the beads for 30 min at 65 degrees C with elution buffer (0.1 M NaHCO3, 1 % SDS). Addition of 350 mM NaCl and incubation for 3 h at 65 degrees C reversed the cross-links before treatment of samples with RNase A for 1 h at 37 degrees C and proteinase K overnight at 65 degrees C. DNA was purified using a PCR-purification kit (Qiagen). ChIP DNA was prepared for sequencing by blunting the DNA with a mixture of T4 DNA polymerase, Klenow DNA polymerase, and T4 PNK (NEB) according to the manufacturer's instructions. dA overhangs were then added and Illumina adapters ligated. Adapter-ligated DNA was subject to 18 cycles of PCR before size selection by agarose gel electrophoresis. Amplified DNA was purified using the Qiaquick gel extraction kit (Qiagen). The purified DNA was quantified both with an Agilent Bioanalyzer and Invitrogen Qubit.
Experimental Factor: immunoprecipitate
H3 Millipore/07-690