title: GSM2481153: Tcell input; Mus musculus; ChIP-Seq
library_construction_protocol: 30x106 T cells were crosslinked with 1% Formaldehyde (10min, RT) and crosslinking reaction was stopped by the addition of glycine to a final concentration of 0.125 M. Chromatin immunoprecipitation assays were performed as described (DeKoter RP et al., 2002) with minor changes. After sonification for 25 Cycles (Bioruptor; 30s on, 30s off at high) chromatin was diluted to 1ml total volume in nuclei-lysis buffer with protease inhibitors and added to 3ml Chip IP buffer (0.01%SDS; 1.1% Triton X-100;1.2mM EDTA; 16.7mM Tris-HCl,pH8.1; 16.7mM NaCl) with protease inhibitors. 10μg antibody were coupled to 80μl Dynabeads Protein G (10004D) for at least 1h at RT and further blocked with sonicated salmon sperm DNA for 30min before washing. Beads and diluted Chromatin were incubated at 4°C over night and washed with 1ml of the following buffers each at RT for 5min with rotation: Buffer I (0.1% SDS; 1% Triton X-100; 2mM EDTA; 20mM Tris-HCL pH8.1; 150mM NaCl), Buffer II (0.1% SDS; 1% Triton X-100; 2mM EDTA; 20mM Tris-HCL pH8.1; 500mM NaCl), Buffer III (0.25M LiCl; 1% NP-40; 1mM EDTA; 10mM Tris-HCl, pH8.1), 2x TE ph 8.0. DNA purification was done according to Ronnie Blecher-Gonen et al., 2013. Library preperation was performed from 2 ng of ChIP DNA using the Kapa Hyper Prep Kit with PCR library amplification (#KK8504, KapaBiosystems) according to the manufactur's protocol. Shortly, ChIP DNA was end-repaired and A-tailed before adapter ligation. After adapter ligation, the ChIP DNA was purified with Agencourt AMPure XP beads (#A63880, Beckman Coulter) and size-selected for 360-600 bp using the Pippin Prep System from Saga Science. The size-selected library is amplified by PCR using the Kapa Hyper Prep Kit (#KK8504,KapaBiosystems) and purified terminally with Agencourt AMPure XP beads (#A63880, Beckman Coulter).
identifiers.namespace: GEO
identifiers.id: GSM2481153