GSM2042281: Thymus Satb1 ChIP-seq, a; Mus musculus; ChIP-Seq
For ChIP-seq using anti-SATB1 (abcam, ab109122), 4x10^7 C57BL/6J thymocytes were freshly prepared and washed with PBS containing 0.5 mM PMSF and were subjected to crosslinking by incubation for 10 min in 5 mM HEPES pH 7.5, 10 mM NaCl, 0.1 mM EDTA, 0.05 mM EGTA, 1% formaldehyde at 23 °C. The reaction was stopped by addition of glycine to 0.125 M. Cells were then immediately washed in ice-cold PBS containing 0.5 mM PMSF and lysed by incubation for 10 min on ice in 1 ml of 50 mM HEPES pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% Glycerol, 0.5% NP40, 0.25% TritonX-100 containing protease inhibitor (Roche). Nuclei were pelleted and lysed by resuspending in 10 mM Tris-HCl pH 8.0, 200 mM NaCl, 1 mM EDTA and 0.5 mM EGTA containing protease inhibitor (Roche). Pelleted chromatin was resuspended in 400 μl of 10 mM Tris pH 8.0, 300 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1% TritonX-100, 0.1% sodium deoxycholate and 0.5% N-laurylsarcosine, and was sonicated using a model XL2000 ultrasonic cell disruptor (MICROSON) so that relatively large fragments (1-10 kbp) were included. Sonicated chromatin was incubated overnight at 4 °C with anti-SATB1 antibody that was preconjugated with magnetic beads (Dynabeads). Immunoprecipitated DNA was then purified after vigorous washing of immunoprecipitates and reversal of crosslinks by overnight incubation at 65 °C in 50 mM Tris pH 8.0, 10 mM EDTA, 1% SDS. Input DNA and ChIP DNA (10-20 ng) were subjected to sonication (Bioruptor, UCD-200) on low power for 20 cycles (30 sec. on and 30 sec. off). Libraries were constructed using the ThruPlex DNA-seq kit (Rubicon Genomics) according to the manufacture's protocol, followed by purification using SPRI beads (Thermo Fisher). Libraries were sequenced to 50 bp read length on a HiSeq2000 (Illumina).
GEO Accession
GSM2042281