GSM8940486: YPD, Cas5, ChIP rep2; Candida albicans; ChIP-Seq
Briefly, cells were cultured in 50 mL of YPD medium until the optical density reached 0.6, in which the calcium ion-treated group was supplemented with 0.2 M calcium and continued to incubate for 1 h. Subsequently, cells were crosslinked with 1% formaldehyde for 15 min at 30°C with gentle shaking, and then quenched by adding 0.125 M glycine for 5 min. After three rounds of cold phosphate buffer (PBS) washing, fungal cells were lysed using glass beads in low-salt buffer with added protease inhibitors. After cell lysis, the lysate was sonicated on ice to fragment the chromatin into fragments of 200 to 500 bp size. After removal of the cellular debris, chromatin fragments were captured using protein A/G magnetic beads by the addition of anti-Myc-Tag monoclonal antibody(Abmart, M20002L) or Normal Rabbit IgG (Cell signaling technology, 2729S). The beads were subsequently washed using buffers of different salt concentrations ( in sequence, low salt buffer, high salt buffer, lithium chloride buffer and tri-ethylenediaminetetraacetic acid (TE) buffer). Subsequently, a buffer containing sodium dodecyl sulfate (SDS) and sodium chloride was added and heated overnight at 65°C to promote the release of DNA fragments. The beads were then treated with RNAase and proteinase K sequentially, the supernatant was centrifuged and purified using a DNA purification kit (Thermo Fisher Scientific, K0702). After the DNA sample passes quality testing, chromatin co-precipitation technology is used to specifically enrich protein-bound DNA fragments. The library preparation process is completed through steps including end repair, A-tailing, sequencing adapter ligation, purification, and PCR amplification. Once the library is constructed, it is first quantified preliminarily using Qubit 2.0 and diluted. The Agilent 2100 system is then employed to detect the insert fragment size of the library. If the insert size meets the expected criteria, the Q-PCR method is applied to accurately quantify the effective concentration of the library, ensuring its quality.