title: GSM5740077: Standard TKS407E Thymus KO 150s Satb1 Abcam 109122 (replica 2); Mus musculus; ChIP-Seq
library_construction_protocol: For standard ChIP-seq, the protocol published by Kitagawa et al. (Nat Immunol 2017, 18, 173-183) was basically followed with minor modifications.   Thymocytes prepared from thymus of Satb1-/-  mice (P13) were cross-linked with 1% formaldehyde for 10 min at room temperature. After quenching cross-linking reaction (0.125M Gly at final), these cells were lysed in buffer (4% SDS, 50 mM Tris-HCl [pH 8.0], 10 mM EDTA, 100 mM NaCl) with Protease inhibitor (Roche) and RNase inhibitor. Cross-linked DNA was sonicated using Branson sonifier (20% power, 10 sec on and 30 sec off for 4-6 cycles) in sonication buffer (0.5% Na-laurylsarcosine, 0.1% DOC, 1mM EDTA, 0.5mM EGTA, 10mM Tris-HCl, 100mM NaCl) with Protease inhibitor and RNase inhibitor to generate fragment size 300-1,000bp. For chromatin-immunoprecipitation, 1 µg of an antibody of choice were added to 10 ~ 20 µg of sonicated solubilized chromatin in 350 µl in dilution buffer (1% Triton X-100, 0.01% SDS, 1 mM EDTA, 15 mM Tris-HCl, 100 mM NaCl) and incubated overnight at 4°C. The chromatin fragments bound to antibodies (after incubation overnight) were captured using 40 µl of protein-A or protein-G Dynabeads (Invitrogen; pre-washed with 0.1% BSA [Fraction V, Sigma), by 4 hrs incubation time. After beads washing and DNA elution from beads, ChIP DNAs was reverse-crosslinked at 65oC overnight, followed by RNase-A treatment and proteinase K treatments. 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). The purified DNAs were subjected to ChIP-seq library construction. 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).