GSM8186999: Kabuki syndrome type 1 replicate 3 (replication cohort); Mus musculus; ATAC-seq
Hippocampal neurons were isolated using a protocol adapted from the INTACT (Mo et al., 2015) and Omni-ATAC (Corces et al., 2017) protocols. Dentate gyrus was microdissected as previously described (Carosso et al., 2019) and placed into ice cold 1xHBS (1x HBS-B, 1M sucrose, 500mM EDTA, 10\% IGEPAL; 1x HBS-B was initially prepared as 6xHBS with 1MCaCl2, 1MMgAc, 1M Tris pH7.8, 1mM BME). Samples were homogenized in a 2mL glass dounce, pestle A until resistance went away (approximately 15 strokes) and pestle B until the tissue looked homogenized (approximately 8 strokes). Samples were then spun through a discontinuous iodixanol gradient. Gradients were made by diluting homogenized sample 1:1 with 50% iodixanol solution for a final concentration of 25% iodixanol (60% iodixanol solution + HBS), and overlayed on a 30%-35%  iodixanol gradient in 15 mL conical tubes (30% iodixanol solution: 1xHBS, 30% iodixanol solution, 160mM sucrose; 35% iodixanol solution: 1xHBS, 35% iodixanol solution, 160mM sucrose). Gradients were spun for 5000g 20min 4' with no break in swinging bucket centrifuge. Nuclei were collected at the 30-35% interface, transferred to a new tube and diluted with 1xHBS. Samples were spun for 10min 4' 1000g, aspirated, then resuspended in 200uL 1xHBS. Nuclei were then stained for NeuN (NeuN-488, EMD Millipore MAB377X) and incubated 30min in dark. DNA was counterstained with Hoechest 33342 and sorted on NeuN+ to collect neurons (to avoid glia). 5000 nuclei were collected for most samples, though some variation was present. Samples were resuspended in tagmentation reaction mix for 30 min (2.5uL TD1, 1X TD Buffer, Illumina Nextera DNA, FC-121-1030; .25uL 1% digitonin, Promega G9441; 1xPBS) gently shaking (300rpm on Eppendorf thermomixer) at 37'. We purified reactions using Zymo DNA Clean and Concentrator-5 kit (Zymo D4013) following manufacturer protocols and eluted with 10.5uL water to recover 10uL. Each reaction was then amplified and indexed per standard ATAC-seq protocol. After indexing and amplification, we purified samples using Select-A-Size purification columns (Zymo D4080) with a cutoff of 150bp to remove adapter dimers to allow for efficient sequencing on patterned flow cells, checked library size on BioAnalyzer using DNA High Sensitivity reagents (Agilent 5067-4626) and determined concentration using Qubit dsDNA HS Assay Kit (ThermoFisher Q32851).