GSM5740059: Urea TKS355A P13 Thymus WT input (replica 1); Mus musculus; ChIP-Seq
GSM5740059
Thymocytes isolated from thymus of Satb1+/+ mice (P13) were cross-linked with 1% formaldehyde for 10 min at room temperature, followed by addition of glycine at 0.125 M for 5 min to quench cross-linking reaction. These cells were washed with 1X PBS, suspended in 1% BSA-PBS and cells were precipitated in a tube.  These cells were re-suspended in lysis buffer (4% SDS, 50 mM Tris-HCl [pH 8.0], 10 mM EDTA, 100 mM NaCl) with Protease inhibitor (Roche) and RNase inhibitor.  The resulting cell lysate was incubated for 30 min at room temperature and loaded on top of 8 M urea followed by ultracentrifugation in a Beckman TL-100 Ultracentrifuge for 4-5 hrs at 45K rpm (181,000g, g=relative centrifugation force or RCF; TLS55 rotor).  Recovered chromatin pellet was sonicated to generate fragment size ranging 300–1,000bp using Branson sonifier (20% power, 10 sec on and 30 sec off for 4-6 cycles) in 300ml of sonication buffer [0.5% Na-laurylsarcosine, 0.1% deoxycholate (DOC), 1mM EDTA, 0.5mM EGTA, 10mM TrisHCl, 100mM NaCl] with Protease inhibitor and RNase inhibitor. Note: urea-purified crosslinked chromatin is highly vulnerable to sonication, therefore, it needs to adjust the sonication condition depending on cell type and/or the size of the precipitate DNA.  The sonicated chromatin with 1% Triton X-100 were dialyzed against dialysis buffer (10 mM Tris-HCl [pH 8.0], 100 mM NaCl, 1 mM EDTA, 5% glycerol), and dialyzed for 16 hrs at 4°C (this dialysis is optional).  The chromatin sample was centrifuged at 15,000g for 5 min at 4°C to remove a trace amount of residual insoluble components. The test-reverse cross-linking of chromatin aliquot (5-10ml of the final volume) was done at 65°C for 2-3 hrs in buffer containing 1% SDS, 100 mM NaCl, 1 mM EDTA, 10mMTris.  Samples were further treated by 100 µg/ml RNase A at 37°C for 1 hr, 200 µg/ml proteinase K at 60°C for more than 3 hrs, followed by DNA purification using QIAquick PCR purification kit (Qiagen), and shearing efficiency was examined by Bioanalyzer 2100 (Agilent Technologies Inc.). The sonicated crosslinked chromatin before immunoprecipitation (input chromatin) was reverse-crosslinked at 65°C for 6 hrs, followed by RNase-A treatment at 37°C for 1 hr and proteinase K treatments for 3 hrs at 60°C. The recovered DNA was further purified with a QIAquick PCR purification kit (Qiagen), with elution in 15µl of Qiagen Elution Buffer (alternatively, Phenol-chloroform extraction and ethanol precipitation).  DNA isolated from input chromatin, at a comparable amount to that isolated from ChIP chromatin, was subjected to library preparation for sequencing. Briefly for library construction, DNA fragments were end-repaired and ligated to indexed sequencing adapters (NEB or Rubicon library kit) and amplified with 8-11 rounds of PCR, depending on the DNA amount.  Using SPRI beads (Beckmancoulter), small fragments (<200bp) were removed from the amplified library. The size-selected library was sequenced to 50bp single-end read lengths on an Illumina HiSeq4000 (Vincent J. Coates Genomic Sequencing Laboratory, UC Berkeley).