GSM5513656: CD14+ Monocytes-CXCL4-ATAC-Donor1; Homo sapiens; ATAC-seq
~0.5-1 million CD14+ human monocytes were collected and centrifuged at 500 × g for 5 min at 4 °C. Omni-ATAC protocol was implemented to obtain ATAC-seq libraries. Briefly, CD14+ monocyte cell pellets were washed once with 1x PBS followed by centrifugation at 500 g for 5 min at 4oC. Cells pellets were lysed in 50 μL of lysis buffer. Cell pellets were gently resuspended in 50 μL of resuspension buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% v/v IGEPAL CA-630, 0.1% v/v Tween-20, 0.01% Digitonin) by pipetting up and down 3x times followed by incubation on ice for 3 minutes immediately followed by the addition of l ml of RSB buffer containing only 0.1% v/v Tween-20 but without 0.1% v/v IGEPAL CA-630 and 0.01% Digitonin. The lysates were centrifuged immediately at 500 × g for 10 min at 4 °C. The supernatants (cytoplasmic contents) were discarded and the resulting nuclear pellet containing intact chromatin was subjected to TN5 mediated transposition. The nuclear pellet was resuspended in a final volume of 50 μl transposase reaction mix (25 μL 2 × TD buffer, 2.5 μL Illumina Tn5 transposase, 16.5 μL 1X PBS, 0.5 μl 10% Tween-20 (final 0.1% v/v), 0.5 μl 1% Digitonin (final 0.01% v/v), 5 μl nuclease-free H2O). The transposition reaction was carried out for 30 min at 37 °C on a thermomixer set at 1,000 rpm. Immediately following transposition, the sample was purified using a QIAGEN MinElute Purification Kit. The transposed DNA was amplified using NEBNext 2x PCR master mix and 1.25 M of ATAC indexing PCR primers, using the following PCR conditions: 72 °C for 5 min; 98 °C for 30 s; and thermocycling at 98 °C for 10 s, 63 °C for 30 s, and 72 °C for 1 min. The PCR was first performed for a maximum of 5 cycles and the optimal number of PCR cycles were determined for each sample to avoid variation among samples due to PCR bias. The libraries were purified using a QIAGEN PCR purification kit followed by a quality control check on a bioanalyzer to check the distribution of the transposed DNA fragments. Libraries were pooled and subjected to high-throughput sequencing on an Illumina Hi-Seq 4000 Sequencer.