GSM7677864: WT, replicate 3, 10X multiome scRNA; Mus musculus; RNA-Seq
GC B cells were isolated from mice spleens at day 10 post-immunization using the Germinal Center B Cell (PNA) MicroBead Kit (Miltenyi Biotec,130-110-479). To improve the quality of single cell ATAC libraries, pre-enriched cells were stained with Zombie Green (BioLegend) and sorted by FACS. Cells were washed in PBS containing 0.04% BSA 2 times. Determine the cell concentration using a Countess II FL Automated Cell counter and viable cell number was counted by the trypan blue dye exclusion method, before proceeding to nuclei isolation. Pellet 100,000~200,000 cells at 300 rcf for 5 min at 4 °C, remove all supernatant and resuspend in lysis buffer (10 mM Tris-HCl, 10 mM NaCl, 3 mM MgCl2, 0.1% Tween-20, 0.1% NP-40, 0.01% Digitonin, 1% BSA, 1 mM DTT and 1 U/µl RNase inhibitor). Incubate for 5 min on ice. After a total of 3 washes, nuclei were resuspended in diluted nuclei buffer (10x Genomics) and nuclei concentration was determined. Proceed immediately to Chromium Next GEM Single Cell Multiome ATAC + Gene Expression User Guide (CG000338). Briefly, 10,000 nuclei/sample were loaded on the Chromium Controller. The 10X libraries were then prepared following the manufacturer's instruction (Next GEM Single Cell Multiome) Chromium Next GEM Single Cell Multiome ATAC + Gene Expression