GSM9136286: FTD hTau sciATAC Drosophila Exp 210624; Drosophila melanogaster; ATAC-seq
sciATAC-seq Flies (5-7 days and 30 days old) were frozen at -80°C and the heads collected using a sieve on dry ice (20-60 heads). The heads were gently sheared open using a Teflon homogenizing stick in 150µl cold Nuclei Isolation Buffer (NIB) (10 mM HEPES-KOH, pH 7.2 [Fisher, Cat. BP310-1], 10 mM NaCl [Fisher, Cat. M-11624], 3 mM MgCl2 [Sigma, Cat. M8226], 0.1% IGEPAL [v/v; Sigma, I8896], 0.1% Tween-20 [v/v, Sigma, Cat. P7949], and 1x protease inhibitor [Roche, Cat. 11836170001]). All contents were transferred to a 1mL Dounce-homogenizer with 300µl of cold NIB and left for 5 minutes. The A (loose) pestle was used 3-4 times to achieve a turbid solution then left to incubate for 15 minutes on ice. The B (tight) pestle was then used 3-4 times until the heads were sheared and left to incubate for 15 minutes on ice. The solution was strained using a 35 µM cell strainer and the nuclei concentration was determined using a hemacytometer.  snRNA-seq Fly heads (from 5-7 day old) were removed and collected into 1xPBS (phosphate-buffered saline) on ice (~20 heads) and gently homogenized using a Teflon homogenizing stick in 150µl of cold Nuclei Isolation Buffer (NIB) (10 mM Tris HCl, pH 7.4, 10 mM NaCl [Fisher, Cat. M-11624], 3 mM MgCl2 [Sigma, Cat. M8226], 0.1% IGEPAL [v/v; Sigma, I8896], 0.5% RNase Inhibitor Murine NEB [M0314S]). The contents were transferred to a 1mL Dounce-homogenizer with 350 µl of cold NIB and left for 5 minutes. The A (loose) pestle was used 4 times to achieve a turbid solution then left to incubate for 15 minutes on ice. The B (tight) pestle was then used 4 times until the heads were sheared and left to incubate for 15 minutes on ice. The solution was strained using a 35 µM cell strainer and spun at 500 rcf at 4°C for 10 minutes. The nuclei were washed twice with 400µl of the NIB without IGEPAL. The nuclei concentration was determined using a hemacytometer. sciATAC-seq Single cells were uniquely labeled using two rounds of combinatorial indexing. The first round utilized transposase to introduce an index into open regions of chromatin. Each 96 well plate used had a unique index oligo pre-loaded into the Tn5. Fresh 4X TAPS buffer was diluted with cold NIB to 1X and used to dilute nuclei to desired concentration of 200 nuclei/µl (132 mM TAPS (N-[Tris(hydroxymethyl)methyl]-3-aminopropanesulfonic acid) pH=8.5, 264 mM potassium acetate, 40 mM magnesium acetate, and 64% dimethylformamide). 10 µl of nuclei solution and 1.5 µl 8µM uniquely indexed transposase was added to each well on ice (Picelli et al., 2014). The 96-well tagmentation plates were then incubated on a thermomixer at 55°C with gentle shaking at 300 rpm for 15 minutes. Once completed, the plates were immediately returned to ice to prevent over-transposition and nuclei lysis. All wells for each strain or experimental condition were pooled on ice. Each condition was pooled separately to limit sorting bias. Nuclei were stained with DAPI (5mg/mL) prior to sorting with the BD FACS Diva software (v8.0.1) on a Sony SH800 FACS machine. Sorting 96-well plates with Transposase Neutralization Buffer (8.5 µl/well) were prepared in advance and placed on ice (0.25 µl BSA [NEB, Cat. B9000S], 0.5 µl 1% SDS, 7.75 µl dH20 per well). Indexed PCR primers were added prior to sorting (2.5 µl of 10 µM i5 indexed PCR Primer and 2.5 µl of 10 µM i7 indexed PCR Primer. For each condition, 15 events per 96-well plate were sorted per PCR plate. After sorting, the transposase was denatured, and nuclei were lysed at 55°C for 20 minutes in SDS Transposase Neutralization Buffer. The plates were spun down and frozen at -20°C. The second index was added via PCR on the BioRad CFX real-time cycler running CFX Manager (v3.1) software. 11.5 µl of PCR Master Mix was added to each well of the 96-well nuclei sorted plates (5 µl 5x KAPA HiFi Buffer (GC Buffer), 0.75 µl 10mM KAPA dNTP mix, 0.5 1U/µL KAPA HiFi DNA Polymerase, 0.25 µl 100X SYBR Green I, 5µl dH2O). The real time PCR was performed with the following setup: 72°C 5min, 15-21 cycles of 98°C 30sec, 63°C 30sec, 72°C 1min, Plate Read, 72°C 20sec. The libraries were stored at -20°C after spin down. 10µl from each PCR reaction was pooled and DNA was concentrated using the Nucleospin Gel and PCR Clean-up kit and size selection using 1X SPRI beads (DNA >200 bp). The DNA concentration was quantified using the Qubit 2.0 fluorometer High-Sensitivity kit and then diluted to 1ng/µl for library fragment size and concentration measures using the Agilent Tapestation.  snRNA-seq The Chromium Single Cell 3ʹ v3 protocol was used for index introduction, library construction and sequencing (Zheng et al., 2017).