title: GSM6436447: 10x scATAC-seq on the Drosophila third instar larvae eye disc; Drosophila melanogaster; ATAC-seq
library_construction_protocol: Wandering third instar larvae were collected and a total of ~30 eye-antennal discs were dissected and transferred into a tube containing 200 µL of ice-cold PBS. The sample was centrifuged at 800 g for 5 minutes, and after removing the supernatant, resuspended in 500 µl of nuclei lysis buffer (10 mM Tris-HCl (pH 7.4), 10 mM NaCl, 3mM MgCl2, 0.1% Tween-20, 0.1% Nonidet P40, 0.01% Digitonin, 1% BSA, and water) and transferred to a dounce homogenizer (Sigma-Aldrich-D8938_2mL). After incubating the sample for 5 minutes on ice, 25 strokes were applied with the loose pestle. The sample was incubated for 10 minutes on ice and after applying 25 strokes with the tight pestle, transferred to a 2 mL tube. The homogenizer and the pestle were rinsed with wash buffer (10 mM Tris-HCl (pH 7.4), 10 mM NaCl, 3mM MgCl2, 0.1% Tween-20, 1% BSA and water), and the solution was also transferred to the 2mL tube. The sample was washed once with wash buffer and resuspended on 50 µl of 1X diluted nuclei buffer (10X Genomics). The nuclei were passed through a 10 µm pluriStrainer (ImTec Diagnostics_435001050) and cell viability and nuclei concentration were assessed by the LUNA-FL Dual Fluorescence Cell Counter. Single-cell libraries were generated using the GemCode Single-Cell instruments and the Single Cell ATAC Library & Gel Bead Kit and ChIP Kit from 10X Genomics, following the protocol provided by the manufacturer. Briefly, the eye-antennal disc nuclei were suspended in 1X diluted nuclei buffer (10X Genomics). About 8,700 nuclei were added in each reaction with a targeted nuclei recovery of 5,000 nuclei. The samples were incubated at 37°C for 1 hour with 10 µl of transposition mix (per reaction, 7 µl ATAC Buffer and 3 µl ATAC Enzyme (10X Genomics)). Following the generation of nanoliter-scale Gel bead-in-EMulsions (GEMs), GEMs were reverse transcribed in a C1000 Touch Thermal Cycler (Bio Rad) programed at 72°C for 5 min, 98°C for 30 sec, 12 cycles of 98°C for 10 sec, 59°C for 30 sec and 72°C for 1 min, and held at 15°C. After reverse transcription, single-cell droplets were broken and the single-strand cDNA was isolated and cleaned with Cleanup Mix containing DynaBeads (Thermo Fisher Scientific). cDNA was then amplified with a C1000 Touch Thermal Cycler programed at 98°C for 3 min, 12 cycles of (98°C for 15 s, 67°C for 20 s, 72°C for 1 min), 72°C for 1 min, and held at 4°C twice. Subsequently, the amplified cDNA was fragmented, end-repaired, A-tailed and index adaptor ligated, with SPRIselect Reagent Kit (Beckman Coulter) with cleanup in between steps. Post-ligation product was amplified with a C1000 Touch Thermal Cycler programed at 98°C for 45 s, 14 cycles of (98°C for 20 s, 54°C for 30 s, 72°C for 20 s), 72°C for 1 min, and hold at 4°C. The sequencing-ready library was cleaned up with SPRIselect beads. Single-cell ATAC-seq (10X Genomics)