title: GSM9065796: DNase-seq_DN3_GATA3_KO_rep1; Mus musculus; DNase-Hypersensitivity
library_name: GSM9065796
library_construction_protocol: The GATA3-fl/fl-CreERT mice were treated with or without tamoxifen for 3 days. To sort DN3 T cells, single cell suspension was prepared from thymus and CD4/CD8 T cells were labeled with unlabeled anti-CD4 and anti-CD8 antibody. BioMag Goat anti-rat IgG beads from QIAGEN were added and bead-bound CD4/CD8 T cells were removed. The remaining cells were stained with linage markers (anti-CD4, CD8a, TCR gamma/delta, CD19, B220, Gr-1, CD11b, CD11c, Nk1.1 and Ter119) and anti-CD3, CD44, CD25. DN3 T subset were sorted as linage-CD3-CD44-CD25+.     The primary fibroblasts were isolated from mice ear in WT and BRG1-AID mice. In brief, the ear was cut into 1 ~ 3 mm pieces, incubated with 0.25% trypsin–EDTA solution for 30 min at 37 °C, neutralized by culture medium with FBS and then plated on 6 well plate with coverslip on it. Fibroblasts will migrate from tissues during the 3 ~ 5-day culture period. After reaching the appropriate confluence, tissues will be removed and primary fibroblasts will be passaged and maintained in DMEM medium (high glucose) containing 10% fetal bovine serum.  For plasmid transfection in fibroblasts, 20 μg plasmid DNA was used for each 10 cm dish using a combination of two commercial reagents, Lipofectamine LTX (Cat. No. 15338100) and FuGENE HD (Cat. No. E2311) with the ratio of 1:1. The auxin was added 24 h later after transfection and incubated for 8 ~ 10 h. Then, the live fibroblasts were sorted for further experiments. DNase-seq assays were performed as described previously. Briefly, 10,000 cells from each group were collected by FACS sorting and fixed in 1% formaldehyde for 10min at RT. The fixation was stopped by glycine and washed twice by PBS buffer. DNase I was added to each sample and incubated at 37 °C for 1 min. Reactions were stopped by adding stop buffer (10 mM Tris-HCl, 10 mM NaCl, 0.1% SDS, 10 mM EDTA) containing 1 μl of 20 mg/ml proteinase K. Samples were incubated at 65 °C overnight, and DNAs were purified by using MinElute reaction cleanup kit following the manufacturer's protocol. DNase-seq libraries were prepared using NEBNext® Ultra™ II DNA Library Prep Kit for Illumina. For fibroblasts, equal amount of human 293T cells were added as spike in before DNase treatment.
identifiers.namespace: GEO
identifiers.id: GSM9065796_r1