GSM5065247: abx27_ctrl-d30-pDC_A069; Homo sapiens; ATAC-seq
Atac-seq was performed on purified innate immune cell subsets immediately after sorting based on the low-input Omni-Atac protocol described before (Corces et al., 2017). In brief, 1,500 – 5,500 cells were washed with ATAC resuspension buffer (10 mM Tris-HCl pH 7.5 [Invitrogen, 15567027], 10 mM NaCl [Invitrogen, AM9760G], 3 mM MgCl2 [Invitrogen, AM9530G], in water [Invitrogen, 10977015]) and supernatant was carefully aspirated, first using a P1000, then a P200 pipette. Next, 10 µL transposition mix (0.5 µL Tn5, 0.1 µL 10% Tween-20, 0.1 µL 1% Digitonin, 3.3 µL PBS, 1 µL water, and 5 µL tagmentation buffer) was added to the pellet and cells were resuspended by pipetting up and down 6 times. Tagmentation buffer was prepared locally by resuspending 20 mM Tris-HCl pH 7.5, 10 mM MgCl2, and 20% Dimethyl Formamide (Sigma Aldrich, D4551-250ML) in water. Cells were incubated at 37C for 30 minutes under constant mixing. After tagmentation, the reaction was cleaned up using the MinElute PCR Purification Kit (Qiagen, 28006) according to manufacturer's instructions. Cleaned DNA was eluted in 21 µL of elution buffer, stored at -20C, and shipped to Active Motif for sequencing library preparation. At Active Motif, tagmented DNA was amplified with 10 cycles of PCR using customized Nextera PCR Primers 1 and 2 (see Key Resource table), and purified using Agencourt AMPure SPRI beads (Beckman Coulter, A63882). Resulting material was quantified using the KAPA Library Quantification Kit for Illumina platforms (Roche, 07960255001), and sequenced with PE42 sequencing on the NextSeq 500 sequencer (Illumina).