GSM1541965: ATAC-seq_excitatory_neurons_rep2; Mus musculus; OTHER
The neocortex was rapidly dissected from 1-2 adult mice on ice, and the INTACT method was used to affinity purify GFP+ nuclei. Approximately 50,000 bead-bound nuclei were transposed in a 50uL volume of 1X TD buffer and 2.5uL Tn5 transposase (Illumina FC-121-1030) for 30 minutes at 37°C, as previously described [Buenrostro J.D. et al. Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position. Nat. Methods 10, 1213-1218 (2013)], with the modification that fragmented genomic DNA was recovered using Buffer QG (Qiagen 28604). Transposed genomic DNA was amplified by five cycles of quantitative PCR. 10% of the PCR was subjected to an additional 20 cycles of SYBR green-based qPCR while the remainder of the sample was left on ice. Analysis of the qPCR data allowed a rough estimate of the number of additional cycles needed to generate product at 25% saturation. Typically, four to seven additional PCR cycles were added to the initial set of five cycles. Amplified DNA was purified on AMPure XP beads, analyzed on an Agilent Bioanalyzer, and sequenced (paired-end) on an Illumina HiSeq 2500 for 101 cycles.