title: GSM6929023: mRNA-Seq 3T3-L1-AD, Cebpa-mRNA-Seq 3T3-L1-AD, rep2; Mus musculus; OTHER
library_construction_protocol: For cultured cells, 1 × 107 cells were cross-linked with fresh 2% formaldehyde for 10 min at room temperature. For adipose tissue, 1g frozen adipose tissue were crushed into powder using a mortar and pestle pre-chilled in liquid nitrogen, and the powder was suspended in PBS and fixed with fresh 2% formaldehyde for 30 min at RT. Crosslinking was quenched with glycine to a final concentration of 0.13 M. Crosslinked cells were lysed for 15 min on ice in 10 mM Tris-HCl pH 8.0, 10 mM NaCl, and 0.2% NP-40 supplemented with protease inhibitor. Nuclei were isolated by centrifugation and by removing the supernatant. The cross-linked chromatin was digested using primary restriction enzyme DpnII (New England Biolabs) and religated by T4 DNA ligase (New England Biolabs) with rotation overnight at 16℃. After reversal of crosslinking and RNA removal, DNA was extracted by phenol/chloroform and purified by ethanol precipitation. Re-ligated DNA was digested using secondary restriction enzyme NlaIII (New England Biolabs) and religated by T4 DNA ligase (New England Biolabs) overnight at 16℃. DNA was extracted by phenol/chloroform and purified using a QIAquick PCR Cleanup kit. The DNA product served as the template for 4C-PCR. 4C PCR was performed with a total amount of input of 3.2 μg and separated into 16 reactions. Two-step PCR strategy was used to construct 4C-seq sequencing library. PCR products were pooled. The PCR products were then run on 2% agarose gels. Smears from 200 to 800 bp were excised on the gel, and unwanted PCR product bands were removed. The DNA was purified using Qiagen PCR cleanup kit and eluted in 10 mM Tris-HCl (pH 7.5). 4C-seq libraries were sequenced using the Illumina NovaSeq 6000 platform (Illumina).