GSM8269035: NAIVE,0.1 ug/ml,ATAC-seq, rep2; Mus musculus; ATAC-seq
Three unimmunized mice were used for B cell isolation. Splenocyte single cell suspensions were generated by crushing spleens in MACS buffer (PBS supplemented with 1% FBS and 5 mM EDTA). Cells were filtered through 70 uM nylon mesh and washed at 1700 RPM, 4 ⁰C, 5 minutes. Pellets were resuspended in RT ACK lysis buffer and incubated RT for 3 minutes, followed by addition of 5-10 volumes of MACS buffer and washing at 1700 RPM, 4 ⁰C, 5 minutes. B cells were then isolated by CD19 Microbeads (Miltenyi Biotec). CD19+ cells were then cultured in the presence of indicated stimuli for 2 h at 37°C before being harvested for ATAC-seq. ATAC-seq was performed using TruePrepTM DNA Library Prep Kit V2 for Illumina® (TD502, Vazyme Biotech). Briefly, around 5,000-10,000 sorted cells in 5 μl were lysed in 50 μl lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2 and 0.1% NP-40) for 10 min on ice. Lysed nuclei were then incubated with the Tn5 transposase and tagmentation buffer at 37 °C for 30 min. Fragmented DNA was purified using 2× VAHTS DNA Clean Beads (Vazyme Biotech) following manufacturer's instructions. PCR was performed to amplify the library for 15 cycles using the following PCR conditions: 72 °C for 3 minutes; 98 °C for 30 seconds; thermocycling at 98 °C for 15 seconds, 60 °C for 30 seconds and 72 °C for 30 seconds; then 72 °C for 5 min. After the PCR reaction, libraries were purified with 1.2× beads and sent for high-throughput sequencing.