GSM5226950: gpeGFP_Ef1aSNAIL_rep2; Mus musculus; ATAC-seq
We used an affinity purification approach to isolate PV neurons from the mouse isocortex, as described in Lawler et al., J Neurosci (2020). Briefly, mice were overdosed with isoflurane, decapitated, and rapidly dissected. Fresh brain tissue was sectioned coronally on a vibratome for precision, and we dissected brain regions relevant to the specific experiment to be processed as separate samples. All dissections took place in cold, oxygenated artificial cerebrospinal fluid (aCSF). After dissection, we isolated nuclei from the samples by 30 strokes of dounce homogenization with the loose pestle (0.005 in clearance) in lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% IGEPAL CA-630). The nuclei were filtered through a 70µm strainer and pelleted with 10 minutes of centrifugation at 2,000 x g at 4°C. We resuspended the nuclei pellets in wash buffer (0.25M Sucrose, 25mM KCl, 5mM MgCl2, 20mM Tricine with KOH to pH 7.8, and 0.4% IGEPAL) for the affinity purification steps. The suspension was incubated with anti-GFP antibody (Invitrogen, #G10362) in wash buffer for 30 minutes at 4°C with end-to-end rotation. After this period, we added Protein G Dynabeads  (Thermo Fisher, cat. 10004D) to the reaction and incubated again for 20 minutes. We separated the Sun1GFP+ fraction from the Sun1GFP- fraction on a magnetic bead rack. Sun1GFP- nuclei in the supernatant were centrifuged at 2000 x g for 10 minutes to pellet nuclei, washed one time, and filtered with a 40µm cell strainer. The Sun1GFP+ nuclei attached to the beads were washed 3-4 times with 800µL wash buffer by resuspending the sample, letting it settle onto the magnet, and removing the buffer. Where cell yield was not a concern, we also performed a large volume wash with 10 mL wash buffer and filtered through a 20µm cell strainer. All nuclei preparations were resuspended in water for the ATAC-seq reaction. For each sample, a small aliquot was stained with DAPI (Thermo Scientific, cat. 62248) and the concentration of nuclei was determined by counting DAPI+ nuclei with a hemocytometer. Next, we combined 50,000 nuclei, 25 µL Tagment DNA Buffer, and 2.5 µL Tagment DNA Enzyme I (Illumina cat. 20034198) into 50 µL total for the transposition reaction. The reaction incubated at 37°C for 30 minutes with 300 rpm mixing. Samples containing beads were gently resuspended every 5-10 minutes throughout the incubation to prevent the beads from staying settled at the bottom. Immediately following incubation, the DNA was column purified with the Qiagen MinElute PCR Purification kit (Qiagen, cat. 28004). Libraries were amplified to ⅓ saturation with dual-indexed Illumina primers. We ensured that samples had the characteristic periodic fragment length distribution of high quality ATAC-seq using Agilent TapeStation assessment. Successful samples were sequenced at low depth on the Illumina Miseq system to determine appropriate library pooling and sequencing depth, then paired-end sequenced for 2 x 150 cycles with the Novaseq 6000.