title: GSM7307978: hILC2, ATAC, rep2; Homo sapiens; ATAC-seq
library_construction_protocol: Human ILC2s were isolated from a TrimaAccel LRS Chamber containing peripheral blood of health donors with the RosetteSep Human ILC2 Enrichment Kit (Stem Cell). ILC2s and pro-inflammatory conditioned ILC2s (pcILC2s) were cultured at 2.25 x 105 cells/mL in aMEM medium supplemented with 20% FBS and 1% Pen-Strep along with 50 ng/ml rIL-7, IL-2, IL-25, IL-4 and rIL-33 for the ILC2s and 50 ng/ml rIL-7, 50 ng/ml rIL-12, IL-2, rIL-1b, and 25 ng/ml rIL-18. After 14-21 days, viability and purity was assessed by flow cytometry and nuclei were isolated by pelleting 25,000 - 200,000 in a fixed-angle centrifuge. Mouse ILC2s were cultured at 2.25 x 10^5 cells/mL for 6 days in complete media (RPMI-1640 supplemented with 10% FBS, 2 mM L-glutamine, 12 mM HEPES, 0.1 mM non-essential amino acids, 1 mM sodium pyruvate, 1% Pen/Strep, and 50 μM 2-mercaptoethanol) and supplemented with 10 ng/ml rIL-7 and rIL-33 (PeproTech), with the media changed every 2 days. For experiments in which cells were generated via cytokine-mediated skewing (pcILC2s), cells were cultured at 2.25 x 105 cells/mL for 48 hours in complete media supplemented with 10 ng/ml rIL-7 and rIL-33 (PeproTech). On Days 2 and 4, the media was replaced with complete R10 containing 10 ng/ml rIL-7, 10 ng/ml rIL-12, 10 ng/ml rIL-1b, 10 ng/ml rIL-15, 10 ng/ml rIL-2, and 5 ng/ml rIL-18. For library preparation for both mouse and human ILC2s, cells were lysed prior to 30 minutes of transposition with an in house Tn5 transposase at 37°C. Immediately following transposition, samples were purified with Zymo Conc & Clean (Genesee) and stored at -20°C prior to library amplification. Fragments were amplified using 1× NEBnext PCR master mix (New England BioSciences) and custom Nextera PCR primers. Full libraries were amplified for five cycles, after which a test aliquot of each sample was taken to test 20 cycles to determine the additional number of cycles needed for the remaining 45 μL reaction. After the additional cycles were complete (average of 5-15 additional cycles), libraries were purified prior to a two-sided Ampure bead (Beckman Coulter) size selection to enrich for nucleosome-free fragments. Fragment size and concentration were determined by TapeStation 2000 and Qubit 4 (Agilent).