Zymo chip dna concentrator columns

ATAC-seq libraries were prepared as previously described^{18 (link),23 (link),52 (link),53 (link)} with approximately 50,000 sorted microglia. Cells were lysed in 150 µl lysis buffer (10 mM Tris–HCl pH 7.5, 10 mM NaCl, 3 mM MgCl₂ and 0.1% IGEPAL CA-630 in water). Resulting nuclei were centrifuged at 500g for 10 min. Pelleted nuclei were resuspended in 50 μl transposase reaction mix (1× Tagment DNA Buffer (Illumina 15027866) and 2.5 μl DNA enzyme I (Illumina 15027865)) and incubated at 37 °C for 30 min. DNA was purified with Zymo ChIP DNA concentrator columns (Zymo Research D5205), eludated with 11 µl of elution buffer, and amplified using NebNext High-Fidelity 2× PCR Master Mix (New England BioLabs M0541) with the Nextera primer Ad1 (1.25 µM) and a unique Ad2.n barcoding primer (1.25 µM) for 8–12 cycles. Resulting libraries were size selected by gel excision to 155–250 bp, purified and single end sequenced using a HiSeq 4000 (Illumina) for 51 cycles according to the manufacturer’s instructions.

50,000 FACS isolated classical monocytes or NK cells were lysed in 50 μl lysis buffer (10 mM Tris-HCl ph 7.5, 10 mM NaCl, 3 mM MgCl2, 0.1% IGEPAL, CA-630, in water) on ice and nuclei were pelleted by centrifugation at 500 RCF for 10 min. Nuclei were then resuspended in 50 μl transposase reaction mix (1x Tagment DNA buffer (Illumina 15027866), 2.5 μl Tagment DNA enzyme I (Illumina 15027865), in water) and incubated at 37°C for 30 min on a PCR cycler. DNA was then purified with Zymo ChIP DNA concentrator columns (Zymo Research D5205) and eluted with 10 μl of elution buffer. DNA was then amplified with PCR mix (1.25 μM Nextera primer 1, 1.25 μM Nextera index primer 2-bar code, 0.6x SYBR Green I (Life Technologies, S7563), 1x NEBNext High-Fidelity 2x PCR MasterMix, (NEBM0541)) for 8–12 cycles, size selected for fragments (160–290 bp) by gel extraction (10% TBE gels, Life Technologies EC62752BOX) and single-end sequenced for 51 cycles on a HiSeq 4000 or NextSeq 500.