GSM7877270: LZGC, D10,ATAC-seq, rep2; Mus musculus; ATAC-seq
10-20 mice were used for cell-sorting in each experiment. 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. Single-cell suspension of splenocytes was stained in MACS buffer containing Fc blocker (supernatant from 2.4G2 cell culture) for 20 min on ice before surface staining with primary antibodies in appropriate concentrations on ice for 25 min. Cells were washed again with 5-10 volumes of MACS, and resuspended in MACS for analysis on BD Aria II. 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.