Optimized assay for transposase-accessible chromatin by sequencing (ATAC-seq) library preparation from adult Drosophila melanogaster neurons

Assay for transposase-accessible chromatin by sequencing (ATAC-seq) is rapidly becoming the assay of choice to investigate chromatin-mediated gene regulation, largely because of low input requirements, a fast workflow, and the ability to interrogate the entire genome in an untargeted manner. Many studies using ATAC-seq use mammalian or human-derived tissues, and established protocols work well in these systems. However, ATAC-seq is not yet widely used in Drosophila. Vinegar flies present several advantages over mammalian systems that make them an excellent model for ATAC-seq studies, including abundant genetic tools that allow straightforward targeting, transgene expression, and genetic manipulation that are not available in mammalian models. Because current ATAC-seq protocols are not optimized to use flies, we developed an optimized workflow that accounts for several complicating factors present in Drosophila. We examined parameters affecting nuclei isolation, including input size, freezing time, washing, and possible confounds from retinal pigments. Then, we optimized the enzymatic steps of library construction to account for the smaller Drosophila genome size. Finally, we used our optimized protocol to generate ATAC-seq libraries that meet ENCODE quality metrics. Our optimized protocol enables extensive ATAC-seq experiments in Drosophila, thereby leveraging the advantages of this powerful model system to understand chromatin-mediated gene regulation.

Nuclei isolation. All equipment and buffers were pre-chilled to be ice-cold. All plastic and glassware used for nuclei isolation was pre-treated with 1% bovine serum albumin (BSA) in phosphate-buffered saline (PBS). Flies were collected into empty bottles and frozen at − 80 °C. Then, the flies were placed in pre-chilled (in a − 80 °C freezer) sieves and agitated for 1 min to separate the heads from the bodies. The fly heads were added to lysis buffer [10 mM Tris-HCl pH 7.5, 10 mM NaCl, 3 mM MgCl 2 , 0.1% Tween-20, 0.1% Nonidet P40 substitute, 0.01% digitonin (ThermoFisher Scientific, Waltham, MA, USA), and 1% bovine serum albumin] in a Dounce homogenizer. The tissue was homogenized with the A pestle until the resistance disappeared. The crude homogenate was passed through a 40 μm filter and homogenized with 15 slow strokes with the B pestle. The crude nuclei were washed with wash buffer (10 mM Tris-HCl, 10 mM NaCl, 3 mM MgCl 2 , 1% BSA, and 0.1%  and centrifuged at 500g for 10 min per wash. The washed nuclei were resuspended in 1 mL wash buffer with 3 mM DAPI for evaluation and sorting.

Fluorescence-activated nuclei sorting (FANS).
Nuclei were evaluated and sorted with a BD FACS Aria flow cytometer (BD Biosciences, San Jose, CA, USA) operated by the University of Utah Flow Cytometry Core facility. Nuclei collected from w* Berlin flies (no GFP) were stained with 3 μM DAPI and used as the GFP-negative control to set the sorting gates. GFP-positive nuclei were collected into 500 μL wash buffer and stored on ice until use for ATAC-seq library prep. Nuclei counts were determined using an internal control bead population (Spherotech, Lake Forest, IL, USA) at 10 6 beads/mL. Briefly, 20 μL of well-mixed beads were added to 200 μL of nuclei suspension and gently mixed. Data was recorded for the bead and cell mixture until at least 10,000 singlet beads were collected. Nuclei were identified based on the forward scatter signal and DAPI intensity. The number of nuclei was calculated as follows: number of nuclei recorded × 10 5 /number of beads recorded = number of nuclei/mL. DNA tagmentation, amplification, and purification. Purified nuclei were centrifuged for 10 min at 500g at 4 °C. The pellet was mixed with 22.5 μL 2× Tn5 tagmentation buffer [20 mM Tris-HCl pH 7.6, 10 mM MgCl 2 , and 20% dimethyl formamide (Sigma Aldrich, St. Louis, MO, USA) in sterile water], 16.5 μL 1× PBS, 0.5 μL Tween-20, 5.5 μL water, and Tn5 enzyme (Illumina, Inc., San Diego, CA, USA). The mixture was incubated at 37 °C for various times and the DNA was purified using a MinElute PCR purification kit (Qiagen, Germantown, MD, USA). Then, the purified DNA was mixed with CD index primers (Illumina) and Phusion High Fidelity PCR MasterMix (New England Biolabs, Ipswich, MA, USA) and was amplified by 1  www.nature.com/scientificreports/ for 5 min and 98 °C for 30 s, followed by 5 cycles of 98 °C for 10 s, 63 °C for 30 s, and 72 °C for 1 min in a C100 thermocycler (BioRad, Hercules, CA, USA). 5 μL of this reaction was removed and used for a qPCR side reaction. The aliquot was mixed with the same CD index primers and SsoFast EvaGreen Supermix (BioRad)  and amplified for 1 cycle of 98 °C for 30 s and 40 cycles of 98 °C for 10 s, 63 °C for 30 s, and 72 °C for 1 min in  an Applied Biosystems 7900HT qPCR instrument (ThermoFisher Scientific). Total fluorescence was calculated from the background-subtracted maximum fluorescence of each sample. The cycle number corresponding to a portion of the total fluorescence was determined (~ 8-10 cycles) and the DNA was amplified for this number of additional cycles. After amplification, the DNA was purified with 0.5× and 1.1× AMPure XP beads (Beckman Coulter, Indianapolis, IN, USA) to remove primer-dimers and high-molecular weight DNA (> 5000 bp). The purified DNA was stored at − 20 °C until further analysis.
ATAC-seq library quality assessment. ATAC-seq library quality was assessed using an Agilent 2200 TapeStation and High Sensitivity D5000 ScreenTape assays (Agilent Technologies, Inc., Santa Clara, CA, USA) at the Huntsman Cancer Institute High Throughput Genomics core facility or the Genome Technology Access Center of the McDonnell Genome Institute at the Washington University School of Medicine (St. Louis, MO, USA).
Next generation sequencing and analysis. ATAC-seq libraries constructed using nuclei isolated from dopaminergic and GABAergic neurons were sequenced on a Novaseq 6000 instrument (Illumina) using 50-bp paired-end reads. The sequencing data was analyzed as previously described 28  Statistical analysis. The data are represented as means ± standard deviation. Comparisons between homogenizer size and fly volume were analyzed by two-way ANOVA with Bonferroni's multiple comparison tests. Freezing time was analyzed by one-way ANOVA using Tukey's multiple comparison tests. Eye color experiments, % read alignment, and fractions of reads in peaks (FRiP) were analyzed using Student's t-tests. Gal4-UAS and washing experiments were analyzed by simple linear regression, and analysis of covariance (ANCOVA). All statistical analyses were performed using GraphPad Prism 9 software (San Diego, CA, USA).

Results
Sample volumes, homogenization, and freezing time. Our goal was to identify the optimal conditions for nuclei isolation, purification, and ATAC-seq library preparation using adult Drosophila. We first asked if the number of flies used for head isolation and the size of the Dounce homogenizer play a role in the number of extracted nuclei that are labeled with DAPI. Using a larger volume of flies increased the number of extracted nuclei (2-way ANOVA, F(1,8) = 15.6, p = 0.004). The size of the Dounce homogenizer used for extraction did not significantly affect the total extracted nuclei if analyzed by 2-way ANOVA (F(1,8) = 2.0, p = 0.20; Fig. 1a), however, with 0.5 mL of flies, more nuclei were isolated with the 7 mL homogenizer (significant by isolated t-test). Thus, we recommend using the larger, 7 mL Dounce homogenizer to maximize nuclei extraction. One advantage to using Drosophila is the availability of sophisticated genetic tools that allows the easy identification of target cells, which can then be isolated by flow cytometry. In our nuclei isolation procedure, live neuronal synaptobrevin (nSyb)-Gal4 flies expressing pan-neuronal GFP-nls are frozen at − 80 °C for 5 min, which is similar to slow freezing, since the flies are not immediately frozen when placed in the freezer, as would occur when snap-freezing in liquid nitrogen. Snap-freezing can decrease GFP fluorescence 34 and damage neurons 19,35 , thus decreasing the number of viable GFP-positive nuclei collected by flow cytometry. We hypothesized that extended freezing at − 80 °C could reduce GFP fluorescence or nuclear viability, so we performed a time-course experiment to examine whether freezing time influences the detection of GFP-positive nuclei by flow cytometry. Five minutes is about the minimum time per sample in which flies can be frozen at − 80 °C and homogenized when handling multiple samples. We observed that GFP fluorescence decreased quickly with additional time on dry ice but reached a plateau at ~ 60% fluorescence in the time analyzed (Fig. 1b, one-phase exponential decay, K = 0.51 min −1 , goodness of fit R 2 = 0.64). Therefore, we suggest that flies should only be frozen briefly (5 min or less) at − 80 °C when used for nuclei isolation and ATAC-seq library preparation.
Flow cytometer specificity and sensitivity. ATAC-seq is prone to mitochondrial DNA contamination 6,18 , which reduces the number of reads aligning to the nuclear genome. Since Drosophila can be used to easily label target nuclei, fluorescence-activated nuclei sorting is a convenient way to isolate nuclei of interest while simultaneously reducing mitochondrial contamination. Sorting GFP+ nuclei using a flow cytometer is dependent on the gating scheme for the detection of green fluorescent signal. Detection schemes for stains usually come at a low gate (less signal required) and a high gate (more signal required). To determine the sorting sensitivity at these gates, we ubiquitously expressed a brighter version of nuclear GFP, UAS-Stinger, using a strong Tubulin-Gal4 driver. At the low gate, we recovered ~ 80% of nuclei from ~ 100,000 DAPI+ nuclei (Fig. 2a,b), while we recovered ~ 12% of nuclei with the high gate (Fig. 2c,d). The data from these experiments suggest that the low gate has six-to seven-fold better sensitivity than the high gate. www.nature.com/scientificreports/ To examine the sorting specificity, we evaluated the number of sorted 'GFP+ nuclei' in w* Berlin flies, which are all GFP-negative. Sorting at the low gate yielded 0.093% 'GFP+' nuclei ( Fig. 3a), while at the high gate, 0.026% of nuclei were 'GFP+' (Fig. 3b). Together, these data suggest that the high gate has fourfold higher specificity and is better at calling true positives. As expected, there is a tradeoff between sensitivity and specificity when using low and high GFP+sorting gates.
Drosophila-specific parameters affect nuclei isolation. Using the high sorting gate, 1 in ~ 4000 nuclei are selected as false-positive. At face value, this is good specificity. However, assuming that a Drosophila head contains > 200,000 nuclei, > 50 of those will be sorted as false positives at the high gate (and > 200 at the low gate). These numbers would significantly contaminate the nuclei sorted from a genotype where Gal4 is expressed in a subset of only hundreds of neurons. The high gate will produce fewer false positives, thus increasing the 2 mL 7 mL 2 mL 7 mL  www.nature.com/scientificreports/ specificity for the Gal4-expression pattern, but the high gate will also reduce the sensitivity and recovery of those Gal4 + neurons. Therefore, we asked whether we could increase the recovery via genetic means by increasing the number of Gal4 or UAS-GFP-nls transgenic constructs to boost GFP expression. We used ics-Gal4 UAS-GFP-nls flies and crossed them to themselves, ics-Gal4, UAS-GFP-nls, or w* Berlin to generate flies with 4, 3, 3, or 2 combined copies of Gal4/UAS insertions, respectively. ics-Gal4 expression includes many neurons and the expression pattern is not different between ics-Gal4/ + hetero-and ics-Gal4 homozygotes 36 . Expanding the copies of Gal4/ UAS insertions increased the number of detected GFP+ nuclei (linear regression, slope ≠ 0, p = 0.0008, low gate; p = 0.001, high gate, Fig. 4a). At both gates, the recovery with 4 transgenes was about 20-fold higher than recovery with 2 transgenes. We therefore recommend that for Gal4-lines with sparse expression patterns, the amount of GFP produced should be increased genetically.
Most Drosophila transgenic insertions are marked with a miniature copy of the white + gene, w +mC . The presence of such a transgene will confer anywhere from pale yellow to red-colored eyes. In addition, the allele w +mC is dosage-sensitive, i.e. the more transgenes in the fly labeled with w +mC , the closer to wild-type red the eye color will be. Since we suggested above that increasing the copy of Gal4 and/or UAS transgenes will increase the recovery of GFP+ nuclei, we next wanted to determine if red-colored eye pigment interferes with sorting GFP+ nuclei. We crossed w -;Cha-Gal4 UAS-GFP-nls males to white-eyed (w − ), or to red-eyed (w + ) virgins and then collected the male progeny. Both of these male genotypes contain 2 w +mC alleles from the transgenes, but one group is wild-type red-eyed (w + ) and the other is orange-eyed (in a wbackground). At the low gate, we recovered almost twice as many 'GFP+ nuclei' (Fig. 4b), suggesting that red eye pigment from w + contributes to the false positive rate. We hypothesized that increasing the number of washes during nuclei isolation would mitigate the false positive rate caused by red eye pigments. Washing the crude nuclei extracts one, three-, or five-times dose-dependently reduced the GFP+/DAPI+ ratio in red-eyed and orange-eyed flies. For the red-eyed flies, we observed the steepest decrease between one and three washes, and with three washes, there was no longer a difference in the GFP+/DAPI+ ratio between red-and orange-eye flies (Fig. 4c,d). We therefore recommend at least three washes, especially when sorting with the low-stringency gate and as more w +mC -marked transgenes are used, since the eye color will approach wild-type red.

Reaction conditions affect library quality.
Published ATAC-seq guidelines are based on mammalian genomes, which are approximately tenfold larger than the Drosophila genome 25,26 . Therefore, the parameters used for the Tn5 transposition reaction (incubation time and enzyme concentration) in mammalian cells may not be appropriate for fly nuclei. To determine the optimal Tn5 reaction conditions, we sorted GFP+ nuclei from  www.nature.com/scientificreports/ fly heads and constructed ATAC-seq libraries. We evaluated the library quality using the guidelines recommended by Buenrostro et al. 37 . We first examined how reaction time affects the fragment distribution by sorting pan-neuronal GFP+ nuclei from nSyb-Gal4 UAS-GFP-nls fly heads. Then, we prepared ATAC-seq libraries using 1× Tn5 enzyme and sample incubation for 5, 23, and 60 min (Fig. 5a). A low reaction time decreased the nucleosome-free peak and increased the proportion of larger fragments up to 1000 bp, suggesting that di-, tri-and multi-nucleosome fragments are over-represented. Incubation for 23 min provided a fragment distribution with more nucleosome-free and fewer mono-and di-nucleosome fragments, while increasing the reaction time to 60 min biased the library toward smaller fragment sizes with relatively little evidence of larger fragments. Thus, for library preparation using Drosophila nuclei, 23 min is the optimal reaction time. We next examined how the Tn5 concentration affects libraries by collecting GFP+ nuclei from tubulin-Gal4 UAS-stinger-nls flies and tagmenting the DNA using 0.1×, 0.3×, 1×, and 3× Tn5 (compared to manufacturer recommendation; Fig. 5b). 1× Tn5 showed the largest peak around 200 bp, with additional skewing towards larger fragments. Reacting DNA using 3× Tn5 reduced both skewness toward larger fragments and the number of nucleosome-free fragments due to over-digestion of DNA [which was size-filtered (150-2000 bp) before analysis, Fig. 5b]. Reducing the Tn5 concentration to 0.1× showed skewness to higher fragments, but overall fewer amplified fragments and reduced overall recovery. We therefore recommend 1× Tn5 as the best balance between recovery of amplified fragments and avoiding over-digestion. Finally, we examined how library amplification affects quality. Like other sequencing library types, ATAC-seq libraries must be amplified to obtain sufficient DNA concentrations for sequencing. Buenrostro et al. 6,37 suggested that ATAC-seq libraries should be amplified to 33% of maximal fluorescence obtained in a qPCR pre-reaction to ensure that sufficient material is present for sequencing, without the introduction of GC and size bias. We hypothesized that the smaller genome size in Drosophila would require more PCR cycles than libraries derived from mammalian nuclei. We tagmented DNA from pan-neuronal GFP+ nuclei from nSyb-Gal4 UAS-GFP-nls fly heads with 1X Tn5 and amplified the tagmented DNA to 25%, 33%, and 50% total fluorescence in a qPCR side reaction (40 cycles) to determine the number of PCR cycles needed for library amplification before sequencing (Fig. 5c). The library that was amplified to 33% total fluorescence had the smallest amount of nucleosome-free fragments and a large proportion of ~ 800-1200 bp fragments, which may correspond to a PCR bubble that arose from depleted PCR reagents, most likely primers 38 . In contrast, the library www.nature.com/scientificreports/ amplified to 25% total fluorescence showed a broad nucleosome-free peak and an even distribution of higher molecular weight fragments, while the library amplified to 50% total fluorescence showed a tall nucleosome-free peak that suggests a bias toward low molecular weight amplification. These results suggest that ATAC-seq libraries derived from Drosophila nuclei should be amplified to 25% of total qPCR fluorescence, though 33%-amplified libraries may also be suitable for sequencing after reconditioning PCR 39 .

Optimized reaction conditions generate ATAC-seq libraries conforming to ENCODE standards. Finally, we combined our optimized parameters (Supplementary File 1) to generate and sequence
ATAC-seq libraries. We drove nuclear GFP expression in dopaminergic and GABAergic neurons and isolated the nuclei after freezing approximately 1 mL (300-400) flies at − 80 °C for 5 min. The crude nuclei extracts were washed three times and sorted by flow cytometry using a high-stringency gate. We collected 65,000 nuclei per sample for each neuron type, which were tagmented for 23 min using 1× Tn5. The resulting libraries were amplified to 25% qPCR fluorescence and analyzed using 50-bp paired end sequencing. We then examined the percent alignment to the genome, the fraction of reads in peaks (FRiP), and fragment length distribution of the sequenced libraries. Both libraries showed high alignment rates, with 79% and 94% alignment in dopaminergic and GABAergic libraries, respectively (p = 0.2; Fig. 6a). Dopamine neuron libraries had 0.37 FRiP, while GABA neuron libraries had 0.40 FRiP (p = 0.8; Fig. 6b), which were both higher than the FRiP values (> 0.3) recommended by the ENCODE consortium for ATAC-seq libraries 40 . Finally, each of these libraries showed the expected nucleosomal banding pattern (Fig. 6c). Together, these metrics were similar to those observed in other ATAC-seq studies using adult Drosophila 16 , which indicates that we generated high-quality libraries and that our protocol returns high-quality sequencing data. To test this hypothesis, we examined ATAC-seq peaks in regions associated with classical neuron markers identified by single-cell RNA-seq 41 . We observed dif-  (7)]. Peaks associated with dopamine-neuron specific genes were much smaller or absent in GABA neurons (Fig. 7a). The peaks corresponding to known GABAergic markers 41 include Gad1, which encodes glutamic acid decarboxylase, which synthesizes GABA, Lim3, and CG14989. Peaks associated with GABA marker genes were significantly more open in GABA neurons than in dopamine neurons [peaks indicated by numbers in Fig. 7b; GAD1: p = 0.0005 (1), 4.42e−08 (2), 0.04 (3), 0.01 (4), and 6.6e−08 (5); Lim3: p = 1.39e−05 (6) and 0.001 (7); CG14989; p = 0.1]. These peaks were much smaller or absent in dopaminergic neurons. These results confirm that our nuclei isolation and library generation protocol yields high quality data, including differential chromatin accessibility in known cell-type markers 41 .

Discussion
Previous studies using ATAC-seq focused on either tumor tissue or white blood cells, though recent studies have investigated the brain and other tissues in mice and humans 7,43 . The initial study describing ATAC-seq was performed in mammalian tissue 6 , and published protocols have been optimized for use in these tissues 5,18 . Drosophila melanogaster is a powerful model organism that is routinely used to study development and human disease models. Because we are interested in Drosophila neurons from the adult brain, we first determined some basic parameters for nuclei isolation. Briefly freezing flies does not reduce GFP+ fluorescence, but longer freezing reduced GFP+ nuclei recovery. Using adult Drosophila-heads or whole flies-for ATAC-seq experiments presents some complicating factors, such as the exoskeleton. While brains can be manually dissected, we used www.nature.com/scientificreports/ undissected heads for nuclei isolation, which provides more efficient throughput and sufficient GFP+ nuclei from populations of few neurons. We homogenized and filtered the homogenate, which eliminated exoskeletal debris, while the lysis buffer disrupted the plasma membrane without disrupting the nuclear membrane 18 . Our isolation procedure allowed us to recover up to 80% of GFP+ nuclei. Thus, our physical extraction protocol allows for efficient and sensitive recovery of GFP-labelled nuclei from adult Drosophila heads. An advantage of using Drosophila for ATAC-seq studies is the ability to genetically label nuclei of interest using the Gal4/UAS system. Thousands of Gal4 lines labeling distinct neurons are available 17 , including ones specific to neurotransmitters 44 , allowing for the specific, reproducible interrogation of only a defined subset of neurons. For ATAC-seq, we recommend flies expressing GFP tagged with a nuclear localization signal, since this www.nature.com/scientificreports/ allows simultaneous collection of target nuclei while reducing contamination from non-nuclear DNA. Several studies showed potential cytotoxic effects from GFP overexpression 45,46 , though GFP localized using an NLS does not show adverse cellular effects 47 . Additionally, GFP-tagged histone proteins do not perturb chromatin structure 48,49 . Thus, nucleus-specific GFP expression likely has minimal effects and is suitable for ATAC-seq. Further, FANS has good sensitivity and specificity, which increases the likelihood of generating high-quality ATAC-seq libraries from sorted nuclei. While we recovered only few false positive nuclei from GFP-negative flies, even a few false-positive nuclei may present a problem when aiming to sort from Gal4 lines with sparse expression. Sorting at the high-stringency gate will produce fewer false positives but will also reduce the sensitivity for collecting true positives. Possible solutions to the issue of low GFP+ nuclei recovery include increasing the numbers of fly heads, increasing the GFP signal, or using an anti-GFP antibody pulldown method (isolation of nuclei tagged in specific cell types, INTACT) 50,51 . We recommend that specific Gal4 and UAS-GFP-nls combinations be tested to determine GFP+ nuclei recovery prior to performing actual ATAC-seq experiments, particularly for sparsely expressing target neurons. The Drosophila genome is ~ tenfold smaller than mammalian genomes, so enzyme-mediated steps during ATAC-seq library preparation may be affected by the amount of available DNA. Tn5 tagmentation in mammalian samples is generally performed using undiluted Tn5 enzyme included in a library preparation kit (1×) for 30 min at 37 °C 37 . Because the amount of accessible chromatin in Drosophila nuclei may be lower than in mammalian nuclei, we investigated the effects of changing the parameters of the enzymatic reactions (Tn5 tagmentation and PCR amplification) on fragment distribution and library quality. Previous studies showed that decreasing the Tn5 concentration in mouse embryonic stem cells only decreases tagmentation efficiency when diluted to 10 nM or lower 18 . Notably, this experiment used homemade Tn5 52 , which may have different kinetics or concentrations than commercially-available Tn5 enzyme. Increasing the tagmentation time decreased library complexity, while decreasing the tagmentation time increased the number of mid-length fragments but did not improve nucleosomal periodicity. We also examined the number of PCR cycles needed to amplify ATAC-seq libraries before sequencing. Protocols for generating ATAC-seq libraries from mammalian tissues call for amplification to one-third the total fluorescence in a qPCR side reaction 37 . Because of the smaller genome size in Drosophila, amplifying ATAC-seq libraries to one-third of the total qPCR fluorescence may introduce GC and size bias. Indeed, amplifying our libraries to one-third qPCR fluorescence introduced a PCR bubble, indicating that the available primers were depleted during amplification 38 , causing amplification to occur using annealed adaptor sequences, which increased the mid-size fragments in the library. While these libraries may be sequenceable after one cycle of reconditioning PCR 39 , libraries with PCR bubbles should be quality-checked to verify that the PCR bubble was resolved before sequencing. Based on these results, we recommend transposition with 1× Tn5 for 23 min, followed by amplification of the ATAC-seq libraries to 25% qPCR fluorescence, which preserves library complexity and does not introduce GC or size bias. One difference between our optimized protocol and   (4), and 6.6e−08 (5); Lim3: p = 1.39e−05 (6) and 0.001 (7). CG14989, Lim3, and hth were described as specific markers using single-cell RNA sequencing 41 Figure 8. Flow chart of ATAC-seq library preparation from Drosophila nuclei. Suggested workflow, which we routinely use to generate ATAC-seq libraries from adult Drosophila. The libraries prepared using the workflow conform to ENCODE guidelines, with typical markers indicating a high-quality library, including nucleosomal banding patterns, high genome alignment rates, and high fraction of reads in peaks (FRiP). (a) Pilot study to determine inputs and parameters for the main experiment. (b) Nuclei isolation and tagmentation reaction. (c) Library amplification and purification. In the workflow, blue boxes indicate procedural steps, green diamonds indicate decision points, purple slanted boxes indicate information that inform steps in the main experiment (dotted lines), and the red box indicates a safe stopping point. On the right of each procedural box is shown the approximate time required for each step. www.nature.com/scientificreports/ the original ATAC-seq protocol is the enzyme used for PCR amplification. While the Phusion enzyme we used has been previously used for ATAC-seq library preparation 53 , the Q5 enzyme listed in the original protocol may be more appropriate for transcription factor footprinting or single nucleotide polymorphism profiling due to its higher fidelity. Using these optimized conditions, we generated ATAC-seq libraries from 65,000 nuclei isolated from Drosophila dopaminergic and GABAergic neurons. These libraries were of high quality, with the expected nucleosomal banding patterns and fragment size distributions. Additionally, these libraries had high alignment rates, further confirming their quality. Therefore, we propose that ATAC-seq libraries from Drosophila tissues should use the following parameters (Fig. 8): freezing at − 80 °C for no more than 5 min, at least three washes, 23-min tagmentation time with 1× Tn5, and amplification to 25% qPCR fluorescence. We also suggest that the library quality should be assessed before sequencing via an electrophoresis-based method 37 , a qPCR-based method 54,55 , or both. This will ensure that the sequenced libraries are high-quality, which will ultimately reduce experimental cost. Our protocol harnesses the powerful genetic tools available in Drosophila to achieve excellent cell-type specificity followed by ATAC-seq, which is used to interrogate the chromatin landscape with unprecedented resolution. Thus, our protocol will enable detailed investigations of the role of chromatin-mediated gene regulation in normal and pathological states. www.nature.com/scientificreports/