title: GSM5100364: ATAC-seq; Homo sapiens; ATAC-seq
library_construction_protocol: i3Neurons were grown on 96-well plates (0.05x106 cells per well). Cells were washed gently with PBS. Then, 100 L of accutase per well was added and removed, and the plate was incubated at 37°C for 5 minutes. 50 μL cold lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% IGEPAL CA-630) was added directly to the well for 10 minutes. Cells were then pipetted 10-20 times to break clumps and centrifuged at 500 × g for 10 minutes at 4°C. The nuclei pellet was resuspended in the Nextera transposition reaction mix (25 μL 2x TD Buffer, 2.5 μL Nextera Tn5 transposase (Illumina, Cat. No. FC-121-1030), and 22.5 μL nuclease free H2O) on ice, then incubated for 30 minutes at 37°C. The tagmented DNA was purified using the Qiagen MinElute kit and eluted with 10 μL Elution Buffer. Following purification, library fragments were amplified using the Nextera index kit (Illumina, Cat. No. FC-121-1011) under the following cycling conditions: 72°C for 5 minutes, 98°C for 30 seconds, followed by thermocycling at 98°C for 10 seconds, 63°C for 30 seconds, and 72°C for 1 minute for a total of five cycles. To prevent saturation due to over-amplification, a 5 µL aliquot was then removed and subjected to qPCR for 20 cycles for calculation of the optimal number of cycles needed for the 45 µL reaction that remained. The number of additional cycles required was determined by plotting linear Rn vs. Cycle and calculating the cycle number corresponding to a quarter of the maximum fluorescence intensity. Adding seven cycles to this estimate was found to yield optimal libraries. PCR reactions were subsequently cleaned with Agencourt AMPure XP beads (Beckman Coulter), run on a 2% agarose gel and a smear of 200-800 bp was cut and gel purified using QIAquick Gel Extraction Kit (QIAGEN). Library concentration was determined with KAPA Library Quantification Kit for Illumina Platforms (Kapa Biosystems). Sequencing was performed on the Illumina Nextseq550 (75 bp paired-end reads).