title: GSM5365491: Female environmental enrichment (EE) cortical neurons - ATACseq - rep3; Mus musculus; ATAC-seq
library_construction_protocol: Animals were euthanized by exposure to isoflurane, and frontal cortices were dissected. Mortar and pestle were used to break up tissue in Phosphate Buffered Saline (ph 7.4, PBS) with cOmplete protease inhibitor cocktail (Roche) and RNaseOUT Recombinant Ribonuclease Inhibitor (ThermoFisher). Protease and ribonuclease inhibitors were present in all downstream steps. Tissue was then fixed using 1% paraformaldehyde (Electron Microscopy Sciences) in PBS for 20 minutes. Fixation was quenched with 1M Tris base (ph 8.0) to a final concentration of 250 mM of Tris. Tissue was washed twice in NF1 buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 5 mM MgCl2, 0.1M sucrose, 0.5% Trixon X-100) to remove residual PFA and lyse cells for nuclear extraction. After washing, pellets were then resuspended in NF1 buffer for 30 rounds of dounce homogenization (Wheaton). After douncing, nuclear pellets were obtained by filtering sample through 70 micron mesh, and one more round of NF1 washing. Nuclear pellets were then resuspended in PBS with 1% Bovine Serum Albumin (BSA, ThermoFisher), and stained with Anti-NeuN antibody conjugated to Alexa Fluor 488 (clone A60, EMD Millipore) at a dilution of 1:2000 for 30 minutes. DAPI stain was also added at a concentration of 1:10,000 to aid in identification of nuclei when cell sorting.  After washing, nuclear suspension was subjected to fluorescence-activated cell sorting (FACS) on a BD FACS Aria II with 70 micron nozzle set to 70 PSI. Nuclei were identified via DAPI and neuronal nuclei were selected using the 488 channel. Sorted cells were immediately used for RNAseq or ATACseq library generation. For RNAseq, Because tissues had been fixed prior to nuclear isolation, RNA was extracted from nuclei using the RecoverAll Total Nucleic Acid Isolation Kit for FFPE Tissue (ThermoFisher). Nuclear RNA was run on an Agilent 2100 BioAnalyzer for quality control (sample statsitics provided in Supplementary Table 10). RNA libraries were generated using the SMARTer Stranded Total RNA-seq for mammalian samples (Takara). Of note, libraries were amplified using random hexamer primers so not enriched for mature transcripts. For ATACseq, ATACseq libraries were generated from sorted neuronal nuclei using the Nextera DNA Library Preparation Kit (Illumina), as described previously{Buenrostro:2013bc}. Briefly, nuclear pellet was resuspended in transposase reaction mix buffer, and transposase. Transposition reaction was performed for 30 minutes at 37 ℃. As described previously{Chen:2016kb}, ATACseq libraries can be generated from fixed nuclei. After transposase reaction, defixation buffer (50 mM Tris-HCl ph8.0, 1 mM EDTA, 0.2 M NaCl, 1% SDS, 5 ng/mL proteinase K) was added to the mixture, which was then incubated at 65 ℃ overnight with agitation at 1000 rpm on a benchtop shaker/heater block. The next morning, the reaction was purified using a Qiagen MinElute kit. Following purification, library fragments were amplified using Nextera PCR primers with cycling and cycling number determination as described in the original ATACseq paper cited above.