title: GSM7791720: AAA_ChIP rep1; Mus musculus; ChIP-Seq
library_construction_protocol: Activated CD4+ T cells that had been transduced with wild-type or mutant FoxP3 were sorted based on Thy1.1 reporter expression. For each sample (5X106 cells), cross-linking was achieved with 1% formaldehyde for 10 minutes. Subsequently, the cells were lysed on ice using RIPA buffer (10mM Tris-HCl pH 8.0, 1mM EDTA, 1% Triton X-100, 0.1% Sodium Deoxycholate, 0.1% SDS, 140 mM NaCl, and 1X proteinase inhibitor). Chromatin fragmentation was achieved using an AFA Focused-ultrasonicator (Covaris M220) for 30 minutes (5% duty cycle, 140W max power, 200 cycle/burst), resulting in DNA fragments ranging from 100 to 200 bp. The sheared material underwent a 10-minute centrifugation at 13,000 rpm at 4°C to clear the solution. The cleared material was then subjected to immunoprecipitation overnight with HA-tag antibody (Cell Signaling, #3724) at 4°C, and Protein G beads (Active motif, #53014) were added for an additional 2 hours. The beads were sequentially washed with various buffers: RIPA Wash Buffer (0.1% SDS, 0.1% Sodium Deoxycholate, 1% Triton X­100, 1mM EDTA, 10mM Tris­HCl pH 8.0, 150mM NaCl), RIPA 500 Wash Buffer (0.1% SDS, 0.1% Sodium Deoxycholate, 1% Triton X­100, 1mM EDTA, 10mM Tris­HCl pH 8.0, 500mM NaCl), LiCl wash buffer (10mM Tris­HCl, pH 8.0, 250mM LiCl, 0.5% Triton X­100, 0.5% Sodium Deoxycholate), and Tris buffer (10mM Tris­HCl, pH 8.5). The chromatin was eluted from the beads using Elution buffer (1X TE, pH 8.0, 0.1% SDS, 150mM NaCl, 5 mM DTT). After elution, the DNA was treated with 1 µg DNase-free RNase (Roche) for 30 minutes at 37°C, followed by treatment with Proteinase K (Roche) for at least 4 hours at 63°C to reverse the crosslinks. The reverse-crosslinked DNA was then purified using SPRI beads (Beckman, #B23318). Library preparations steps, including end repair, A-base addition, adaptor-ligation, and PCR amplification, were carried out to prepare the ChIP-seq library for each sample. The libraries were made by NEBNext® Ultra™ II DNA Library Prep Kit (Illumina) according to the manufacturer's instructions and submitted to Novogene for NGS with PE150.