title: GSM6365600: lactate treatment, anti-H3K18la IP (PTM-1406, PTM BIO), biol rep 1; Mus musculus; ChIP-Seq
library_construction_protocol: Cells were fixated with formaldehyde and then harvested and washed two times with ice cold PBS, then resuspended in 1 ml ChIP lysis buffer I (5 mM PIPES pH 8.0, 85 mM KCl, 0,5% (v/v) NP40) and incubated on ice for 20 min. The pellets were then incubated in 1 ml ChIP lysis buffer II (10 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% (v/v) NP40, 1% (w/v) sodium deoxycholate, 0.1% (w/v) SDS, 1 mM EDTA) on ice for 10 min. Chromatin was sheared using an ultrasound desintegrator. Pre-clearing was performed with 100 μl of IgG-linked blocked protein A sepharose (45 min, 4°C, rotator). A 1 % aliquot of each sample was taken as input. Per 2 μg antibody, 220 μl of soluble chromatin was used and immune precipitation was performed. 50 μl of blocked protein A sepharose per sample was added and incubated for 1 h at 4°C. The beads were then washed with wash buffer I (20 mM Tris pH 8.1, 150 NaCl, 1% (v/v) Triton X-100, 0.1% SDS, 2mM EDTA), wash buffer II (20 mM Tris pH 8.1, 500 mM NaCl, 1% Triton X-100, 0.1 SDS, 2 mM EDTA) and wash buffer III (10 mM Tris pH 8.1, 250 mM LiCl, 1% NP40, 1% sodium deoxycholate, 1 mM EDTA) and kept on ice. Afterwards, the samples were washed with Tris buffer (Qiagen EB). Elution was performed by adding 200 μl elution buffer (0.1 M NaHCO3, 1% SDS) and then the samples were incubated for 20 min on a shaker. The samples were then de-crosslinked by adding 16 μl of de-crosslinking buffer and incubated at 65°C overnight. 2.2 ml of binding buffer was added and then the samples were run over a DNA binding column (Qiagen). Elution was performed with 2 x 30-50 μl elution buffer EB. MicroPlex Library Preparation Kit v2