GSM7986306: Urea TKS400G P13 Thymus KO CTCF; Mus musculus; ChIP-Seq
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), shearing efficiency was examined by Bioanalyzer 2100 (Agilent Technologies Inc.). For chromatin-immunoprecipitation, 1 µg of an antibody of choice were added to 10 ~ 20 µg of sonicated solubilized chromatin in 350 ml in dilution buffer (1% Triton X-100, 0.01% SDS, 1 mM EDTA, 15 mM Tris-HCl, 100 mM NaCl) and incubated overnight with rotation at 4°C.  At least three chromatin samples were prepared for ChIP-seq from independent experiments to confirm the results. The chromatin fragments bound to antibodies (after incubation overnight) were captured using 20 µl of protein-A, protein-G or protein AG Dynabeads (Invitrogen; pre-washed with 0.1% BSA [Fraction V, Sigma), by 4 hrs incubation time at 4°C.  Dynabeads bound to chromatin fragments were washed twice with Binding buffer (10 mM Tris, pH8.0, 1 mM EDTA, 1 mM EGTA, 0.5% Triton X-100, 0.1% DOC, 100 mM NaCl), twice with LiCl buffer (100 mM Tris [pH 8.0], 1 mM EDTA, 250 mM LiCl, 0.5% NP-40, and 0.5% DOC).  The tubes were changed and washed twice with 0.1M NaCl-TE buffer. The immunoprecipitated samples were eluted from Dynabeads by elution buffer (10 mM Tris-HCl [pH 8.0], 1 mM EDTA, 100 mM NaCl and 1% SDS) at 65oC for 20 min.  After elution, ChIP DNAs was reverse-crosslinked at 65oC 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). Briefly for library construction, ChIP 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).