Illumina HiSeq 4000 sequencing; ATAC-seq examining chromatin accessibility of mouse splenic IL-10+ and IL-10-CD19+CD21hiCD24hi B cells
ATAC-seq examining chromatin accessibility of mouse splenic IL-10+ and IL-10-CD19+CD21hiCD24hi B cells
Antigen induced arthritis was induced in VertX mice. Spleens were collected day 7 post intra-articular injection and splenocytes were isolated. IL-10+ and IL-10- CD19+CD21hiCD23hiCD24hi and IL-10 CD19+CD21loCD24lo (follicular) cells were isolated by cell sorting. 25,000 cells were collected per sample for DNA preparation. After sorting, 25,000 were washed with 1xPBS (10% FCS). The cell pellet was prepped for sequencing by using the Nextera DNA library preparation kit (Illumina). Briefly, 10µl nuclease free water, 12.5µl 2x Transposase buffer, 2µl transposase and 0.25µl digitonin (0.05%) per reaction were added to the cell pellets. Cells were incubated at 37°C for 30 minutes. DNA was then purified using a MinElute PCR purification kit (Qiagen), according to manufacturer's instructions. Following DNA purification, 1µl of eluted DNA was used in a qPCR reaction to estimate the optimum number of amplification cycles. Library amplification was performed using custom Nextera primers and was followed by solid phase reversible immobilization (SPRI) size selection to exclude fragments larger than 1,200bp.
Experimental Factor: cell type
transitional stage B cell