GSM5329058: ATAC-seq_ATAC69_C3_YD20_ATMAPTX_dKO_PurkinjeCell; Mus musculus; ATAC-seq
Nuclei were isolated according to Xu et. al., (2018), via minor modifications. Isolated  pooled cerebella tissue from 4 wild type mice were collected and snap frozen using liquid  nitrogen, and then thawed on ice for 30min. Cerebella were then transferred to 5ml of  homogenization buffer (0.25 M sucrose, 150 mM KCl, 5 mM MgCl2, 20 mM Tricine-KOH  pH 7.8, 0.15 mM spermine, 0.5 mM spermidine, 5 units EDTA-free protease inhibitor  cocktail, 1 mM DTT, 20 U/mL Superase-In RNase inhibitor, 40 U/mL RNasin ribonuclease  inhibitor) and homogenized by 30 strokes with a glass dounce homogenizer.  Subsequently, 4.6 ml of a 50% iodixanol solution (60% Iodixanol/Optiprep, 900 mM KCl,  30 mM MgCl2, 120 mM Tricine-KOH pH 7.8, 0.15 mM spermine, 0.5 mM spermidine, 4  units EDTA-free protease inhibitor cocktail, 1 mM DTT, 20 U/mL Superase-In RNase  inhibitor, 40 U/mL RNasin ribonuclease inhibitor) was added to tissue homogenates.  Tissue homogenates were then added to a cushion of 2 ml of 27% iodixanol solution in  the bottom of 13.2 ultraclear ultracentrifugation tube (Beckman Coulter; #41121703).  Ultracentrifugation was performed at 10,000 rpm for 45 min at 4°C to isolate nuclei in a  swinging bucket rotor (SW41) in a Beckman Coulter XL-70 ultracentrifuge.  Nuclei pellets were carefully resuspended with 500 ml of homogenization buffer  and fixed with 1 % formaldehyde for 8 min at room temperature. Fixation was stopped by  adding 0.125M glycine for 5 min. Then, fixed nuclei were centrifuged at 1000g, 4 min and  supernatant were discarded. Pellets were washed twice with wash buffer (PBS, 0.05%  TritonX-100, 50 ng/mL BSA, 1 mM DTT, 10 U/uL Superase-In RNase Inhibitor). Nuclei were blocked with blocking buffer (PBS, 0.05% TritonX-100, 50 ng/mL BSA, 1 mM DTT, 10 U/uL Superase-In RNase Inhibitor, and 50 ng/mL BSA) for 30 min. Then, nuclei were incubated with NeuN antibodies (Mouse; 1:250; Millipore, MAB377) and ITPR1 antibodies (Rabbit; 1:500; Invitrogen, PA1-901) for 16hrs at 4°C. Next day, nuclei were washed 3 times with wash buffer and secondary antibodies (1:500) for 30 min at room temperature. Alexa 594 [mouse; ab150116] and Alexa 488 [rabbit; ab150077] were used secondary antibodies. Right before flow cytometry sorting, nuclei were stained with 20uM final concentration of DyeCycle Ruby (Invtrogen; V10273) for 10min at room temperature. Cell type specific nuclei were independently collected using a BD Biosciences FACS Aria cell sorter, with excitation at 488nm, 561nm and 640nm. Gating on single nuclei using width  versus area signals for Dye Cycle Ruby was followed by gating of cell type specific nuclei based on NeuN and ITPR1 signals. Flow sorted granule and Purkinje cell nuclei (5,000 each) were incubated for 10 min in nuclei lysis buffer (10 mM Tris-Cl pH 7.5, 10 mM NaCl, 3 mM MgCl2, 0.05% NP-40), centrifuged for 5 min at 500g and resuspended in 50 ul of transposition reaction mix (25ul of 2xTD buffer, 2.5 ul of Nextera TDE1 enzyme [Illumina; #20034197], and 22.5ul nuclease-free water). The transposition reaction was incubated at 37°C for 30 min with mild shaking. Then, 200ul of reverse-crosslinking solution (50 mM Tris–Cl, 1 mM EDTA, 1% SDS, 0.2 M NaCl, 5 ng/ml proteinase K) was added to transposition reactants for overnight incubation at 65°C in a Thermomixer (Thermo Fisher) with shaking at 800rpm. DNA was then purified with QiaQuick MinElute columns (Qiagen; #28206). and amplified by PCR containing Q5 High Fidelity Polymerase (NEB; M0491S) for 12 cycles with ATACseq barcoded primers. PCR reaction conditions were 72°C for 5 min, 98°C for 30s, 12 cycles of (98°C for 10s, 63°C for 30s, and 72°C for 1 min), 65°C for 5 min, with a hold at 4°C. Amplified libraries were purified and size selected with AMPure XP beads (Beckman Coulter) and then sequenced on an Illumina HiSeq 4000, yielding 100bp paired-end reads.