OEX020247
|
OEX_weifen_2208091019 |
Genomic |
0.15 g of PFC tissue was grinded into powder on dry ice and cross-linked in 1% formaldehyde for 10 min at room temperature. Then, the reaction was quenched by adding glycine, and the tissue was further homogenized in a glass douncer. Cell pellets were lysed successively in Farnham Lysis buffer (5mM PIPES pH 8.0, 85mM KCl, 0.5% NP-40) and Lysis buffer (1% SDS, 10mM EDTA, 50mM Tris-HCl, pH8.0) on ice. Nuclei were sonicated for 5 seconds at 15% power of Misonix Q700 for 90 times with 10 seconds refractory period. The supernatant was diluted in Dilution buffer (1.1% Triton X-100, 0.25% sodium deoxycholate, 1.2mM EDTA, 167mM NaCl, 16.7mM Tris-HCl, pH8.0) and transferred to the Protein A/G magnetic beads which were pre-incubated with H3K4me3 (Millipore, 07-473) or H3K27ac (Active Motif, 39133) antibody in PBS with 0.5% BSA at 4 °C for at least 4 h, and rotated overnight at 4 °C. Beads were then washed sequentially with Low salt wash buffer (0.1% SDS, 1% Triton X-100, 0.25% sodium deoxycholate, 1mM EDTA, 150mM NaCl, 50mM Tris-HCl, pH8.0), High salt wash buffer (0.1% SDS, 1% Triton X-100, 0.25% sodium deoxycholate, 1mM EDTA, 500mM NaCl, 50mM Tris-HCl, pH8.0), LiCl wash buffer (500mM LiCl, 1% NP-40, 1% sodium deoxycholate, 100mM Tris-HCl, pH8.0), TE buffer, and eluted with Elution buffer (1% SDS, 0.1M NaHCO3) at 65°C. After adding NaCl to final 200mM, chromatin in the supernatant was reverse crosslinked overnight at 65 °C, and then treated with RNase A (final concentration of 0.1 μg/μl) for 0.5 h at 37 °C and proteinase K (final concentration of 0.4 μg/μl) for 1 h at 55 °C. DNA was extracted with QIAquick MinElute PCR Purification Kit (Qiagen, 28104). Genomic DNA without the immunoprecipitation was used as an input control. Sequencing library was prepared by using Illumina Truseq ChIP Sample Prep Kit (Illumina, USA). The pooled libraries were sequenced on Illumina HiSeq 2000 platform using the 100-bp singled-ended sequencing protocol. |
2022-08-09 |