GSM1565734: DC_MTB (ATAC-Seq); Homo sapiens; OTHER
DNA from infected and non-infected DCs was extracted using the PureGene DNA extraction kit (Gentra Systems). To prepare nuclei, we spun 100,000 cells at 500g for 5 min, which was followed by a wash using 50 μL of cold 1× 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 MgCl2 and 0.05% IGEPAL CA-630). Immediately after lysis, nuclei were spun at 500g for 10 min using a refrigerated centrifuge. Immediately following the nuclei prep, the pellet was resuspended in the transposase reaction mix (25 μL 2× TD buffer, 2.5 μL transposase (Illumina) and 22.5 μL nuclease-free water). The transposition reaction was carried out for 30 min at 37 °C. Directly following transposition the sample was purified using a Qiagen MinElute kit. Following purification, we amplified library fragments using 25 uL of 2X NEBnext PCR master mix, 0.3 uL of 100X SYBR Green I, 2.5 uL each of Nextera primer index 1 (i7) and 2 (i5), and 10 uL of the transposed DNA, in a final volume of 50 uL. We used 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. To reduce GC and size bias in our PCR, we monitored the PCR reaction using qPCR in order to stop amplification before saturation. To do so, we amplified the full libraries for four cycles, after which we took an aliquot (5 ul) of the PCR reaction and added 10 μl of the previous PCR cocktail. We ran this reaction for 19 cycles to determine the additional number of cycles needed for the remaining 45 uL reaction. Libraries were amplified for a total of 9 cycles. The libraries were purified using a Qiagen MinElute kit in 20 μL. Quality of the libraries was verified on a polyacrylamide gel and Bioanalyzer. Libraries were then quantifed by qPCR using the KAPA Library Quantification Kit.