GSM6304242: J2KO_EpiLC_4C_Tbx3; Mus musculus; OTHER
GSM6304242
Circularized chromosome conformation capture (4C) assays were performed using a modified published approach(van de Werken et al., 2012; Wang et al., 2019). Briefly, 10 million detached ESCs or EpiLCs were fixed with 1% final formaldehyde for 10 minutes, and quenched with final 0.125 M glycine. The cells were lysed in the 30 ml lysis buffer (50 mM Tris pH 7.5, 150 mM NaCl, 0.5% NP-40, 1% Triton, 5 mM EDTA, and  proteinase inhibitors: 2 μg/mL Aprotinin, 1 μg/mL Leupeptin, 1mM PMSF ) with rotation at 4 °C for 45  minutes. Isolated nuclei were then fully digested by restricted enzyme DpnII overnight digestion followed by overnight ligation with 50 U T4 DNA ligase at cool room temperature (18~20  °C) . Ligated circular DNA were purified after de-crosslinking incubation with proteinase K at 65 °C for overnight, followed by further incubation with Rnase A at 37  °C for 1 hour . The purified  DNA was further digested with restricted enzyme NlaIII and then ligated again and purified. The 4C libraries were amplified with the viewpoint-specific primers with inverse PCR using Roche Expand Long Template PCR System. For each viewpoint, at least 8 PCR reaction products were pooled to enhance the library complexity. The 4C PCR products were purified using Qiagen Quick PCR purification Kit. Tbx3 promoter viewpoint primers are AAGGGAAGAAGCTGCAGATC (Reading primer) and TGAAGGGAGCCCCACATG. 4C-seq library preparation was performed using the NEBNext Ultra II DNA Library Kit for Illumina (E7645) according to the manufacturer's protocol. Illumina NextSeq 500, 75-bp single end