title: GSM9116556: NeuN+ ATAC-seq, Rabbit, rep2; Oryctolagus cuniculus; ATAC-seq
library_name: GSM9116556
library_construction_protocol: After centrifugation to remove sucrose gradient, nuclei were labeled by resuspending the pellet in 1 mL blocking buffer with NeuN-488 antibody (Millipore, cat. no. MAB377X) at 1:4,000 and 4,6-diamidino-2-phenylindole at 1:20,000 for at least 30 minutes at 4°C with rotation. Nuclei were then centrifuged at 500 x g for 5 minutes at 4°C. The supernatant was removed the pellet was resuspended in 500 μL cold 1% BSA in PBS with RNase Inhibitor. Nuclei were then filtered through a 35 μm filter, washing the filter with an additional 100 μL 1% BSA in PBS. Nuclei were then sorted using a Sony MA900 device with a 70-μm nozzle and pressure not exceeding a pressure setting of 7. Gates were set to capture those populations that were positive for 488 signal (NeuN+). Both the NeuN+ and NeuN- populations were collected and held at 4°C . Purity of selected samples was typically >95% based on reanalysis of sorted samples. Approximately 100,000 NeuN+ nuclei were isolated from cortex tissues and used for ATAC-seq library preparation as per standard protocols (PMID: 24097267, PMID: 25559105, PMID: 28846090) with the following modifications. Briefly, 50 μL of cold 5x lysis buffer was added to sorted nuclei to reach a final concentration of 1x (1- mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% NP-40) and incubated for 20 min on ice followed by centrifugation for 10 minutes. The transposition reaction was incubated for 30 minutes at 37°C with agitation at 1000 rpm (Illumina Tagment DNA Enzyme and Buffer kit, cat. no. 20034198). After column clean up via the QIAGEN MinElute Reaction Cleanup kit (QIAGEN, cat. no. 28204) and PCR amplification of libraries, an additional clean up with AMPure XP beads (0.8X ratio) was performed with two 80% ethanol washes before quantification using a DNA High-Sensitivity chip on a 2100 BioAnalyzer (Agilent). Libraries were sequenced paired-end with the Illumina dual indexes on a NovaSeq X Plus 10 B 300 cycle flowcell targeting 100 million reads per sample.
identifiers.namespace: GEO
identifiers.id: GSM9116556_r1