GSM5101111: OT-I ATAC-seq 120 min. N4 peptide rep1; Mus musculus; ATAC-seq
Samples of stimulated CD45.2+ OT-I cells and CD45.1+ wildtype splenocytes mixtures were stained with CD8a and CD45.2 antibodies and 7-AAD and OT-I cells were sorted (BD FACSAria) into 100% FBS and pelleted. Precisely 50,000 stimulated OT-I T cells were FACS sorted at the same time as RNA-seq samples and pelleted in 100% FBS. ATAC-seq libraries were generated similarly as described in Buenrostro et al., 2013. Cell nuclei were isolated and transposed with 8 μL Tn5 (Illumina Nextera) at 37 ̊C for 60 minutes. DNA fragments were isolated with a Clean and Concentrator Kit (Zymo) and then Illumina barcoded and amplified with NEBNext High-Fidelity 2x PCR Master Mix (New England Biolabs). A portion of the reaction was performed as a separate qPCR reaction to determine ideal cycle number. Samples were then size selected with SPRI beads to include fragments up to 450 bp, ensuring to maintain small (<200 bp), nucleosome-free fragments. Quality of final ATAC-seq libraries was assessed with fragment analysis and Qubit analysis.