title: GSM5100324: ATAC_Isl5_High-dose_dex_24hr; Homo sapiens; ATAC-seq
library_construction_protocol: Islet samples were centrifuged at 500xg for 3', washed twice in HBSS, and resuspended in nuclei permeabilization buffer. Islets were homogenized using a chilled glass dounce homogenizer and incubated on a tube rotator for 10' before being filtered through a 30uM filter and centrifuged at 500xg in a 4C microcentrifuge to pellet nuclei. Nuclei were resuspended in Tagmentation Buffer (Illumina) and counted using a Countess II Automated Cell Counter (Thermo). ~ 50,000 nuclei were transferred to a 0.2mL PCR tube and volume was adjusted to 22.5uL with Tagmentation Buffer. 2.5uL TDE1 (Illumina) was added to each tagmentation reaction and mixed with gentle pipetting. Transposition reactions were incubated at 37C for 30 minutes. Tagmentation reactions were cleaned up using 2X reaction volume of Ampure XP beads (Beckman Coulter) and eluted in 20uL Buffer EB (Qiagen). 10uL tagmented DNA was used in a 25uL PCR reaction using NEBNext High-Fidelity Master Mix (New England Biolabs) and Nextera XT Dual-Indexed primers (Nextera). Final libraries were double size selected using Ampure XP beads and eluted in a final volume of 20uL Buffer EB.