GC B cells FACS isolated by the new three population strategy were defined as follows. Total GC B cells were defined as B220+GL7+CD95+, then subsets were defined based on CXCR4 and CD83 expression. Proliferative DZ B cells (DZp) were defined as CXCR4+CD83+ (also described as gray zone [GZ] B cells), differentiating DZ B cells were CXCR4+CD83-, and LZ cells were defined as CXCR4-CD83+ GC B cells.
Sequenced DNA Library
library_strategy
ATAC-seq
library_source
GENOMIC
library_selection
other
library_construction_protocol
To prepare Nuclei, respective flow sorted mouse GC B cell subsets (100,000) were centrifuged at 500g for 5 min, which was followed by a wash with ice-cold PBS and centrifugation at 500g for 5 min. Cells were lysed using cold lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2and 0.1% IGEPAL CA-630). Immediately after lysis, nuclei were spun at 500g for 10 min at 4oC and supernatant removed. The nuclei pellet was resuspended in the transposase reaction mix (25 μL 2X Tagment buffer, 2.5 μL Tagment DNA enzyme (Illumina, FC-121-1030) and 22.5 μL nuclease-free water). The transposition reaction was carried out at 37 °C for 30 min. Directly following transposition the sample was purified using a Qiagen MinElute kit. Following purification, we amplified library fragments using Nextera PCR Primers (Illumina Nextera Index kit) and NEBnext PCR master mix (New England lab, 0541) for a total of 10-12 cycles. The libraries were then purified using a Qiagen PCR cleanup kit. The amplified, adapter ligated libraries were size selected using Life Technologies' E-Gel® SizeSelect™ gel system in the 150-650bp range. The size-selected libraries were quantified using the Agilent Bioanalyzer and via q-PCR in triplicate using the KAPA Library Quantification Kit on the Life Technologies Step One System. Libraries were sequenced on the Illumina NextSeq 500 system to generate 75-100M, 42bp paired end reads.