GSM7968376: eGFP_Male_2; Mus musculus; ATAC-seq
Animals were sacrificed by cervical dislocation and bilateral ventral hippocampi were rapidly dissected then flash frozen in liquid nitrogen. Total nuclei were extracted using sucrose gradient centrifugation. Neuronal (NeuN+) nuclei were isolated by fluorescence-activated nuclei sorting with anti-NeuN antibody conjugated to AlexaFluor 488 on a FACSAria instrument. Following FANS, nuclei were pelleted and resuspended in 50 µL of the transposase reaction mix including 25 µL of the 2xTD reaction buffer, 5 µL of transposase enzyme (Illumina Tagment DNA TDE1 kit, 20034197). We also included additional components: 16.5µl 0.1M PBS, 0.5µl 1% digitonin, and 0.5 µl 10% Tween-20, which were previously shown to enhance transposition efficiency. Transposition occurred at 37°C for 30 minutes and transposed DNA was purified using the MinElute PCR Purification Kit (Qiagen, 28004). Indexing libraries using Nextera i5 and i7 indexed amplification primers (Illumina XT index kit, 15055290) occurred alongside library amplification with NEBNext High-Fidelity PCR Master Mix (New England Biolabs, M0541S). The PCR reaction was carried out with the following conditions: 1 cycle of 72°C for 5 minutes and 98°C for 30 seconds; followed by 10 cycles of 98°C for 10 seconds, 63°C for 30 seconds, and 72°C for 1 minute. Amplified libraries were then purified using the MinElute PCR Purification Kit. Library quality was then assessed using the Bioanalyzer High-Sensitivity DNA Assay. The ATAC-seq libraries were quantified by Qubit HS DNA kit and by qPCR prior to sequencing.