biostudies-arrayexpressJuan VaquerizasDrosophila melanogasterhttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-14477Low-input Micro-C (Pico-C) was performed on hand-sorted PCNA-eGFP embryos at interphase from nuclear cycles (NCs) 9 to 14, including double knockdowns of Zelda and GAF and their respective controls (NC14), as well as embryos injected with α-amanitin or water (NC14). ATAC-seq was performed on double knockdowns of Zelda and GAF and their respective controls. RNA-seq was performed on α-amanitin- and water-injected embryos.biostudies-arrayexpressNucleic Acid Extraction - Pico-C nucleic acid extraction was conducted as in Hsieh et al. 2020 and Ing-Simmons et al., 2022 with modifications. At nucelar cycle 14 (NC14) approximately 10 embryos were used per library for a total of 40,000-60,000 nuclei per experiment. Embryos were crushed in the eppendorf tube with metal pestles using 500uL buffer MB1 (50mM NaCl, 10mM Tris, 5mM MgCl2, 1mM CaCl2, 0.2% NP-40, 1X PIC). Nuclei were then bound to Concanavil-A (Biomag) beads, and the remainder of washes were carried out on magnets. Chromatin was digested with a pre-determined amount of Micrococcal Nuclease (Worthington Biochem) to yield ~70% monomer vs ~30% dimer. Size selection was not carried out via gel-extraction but done with AMPure XP (Beckman Coulter) beads after library amplification.Library Construction - Pico-C library construction was conducted as in Hsieh et al. 2020 and Ing-Simmons et al., 2022 with minor modifications. Total DNA was carried over into the final library construction phase, and size selection for the appropriate di-nucleosomal band (350-500bp) was carried out with a two-sided cleanup using AMPure XP (Beckman Coulter) beads.Sequencing - Libraries were pair-end sequenced on Illumina NovaSeq (2x150 bp).Sample Collection - For ATAC-seq on Jabba controls and knockdowns, single Drosophila embryos were collected, staged (nuclear cycle 14), and dechorionated using 50% bleach. Individual embryos were transferred into chilled lysis buffer, manually homogenized under a dissecting microscope, and nuclei were pelleted by centrifugation.Sequencing - RNA-seq libraries were sequenced on an Illumina NextSeq 2000, generating a minimum of 80 million paired-end 60 bp reads per sample.Nucleic Acid Extraction - For library construction, samples were homogenized using a metal pestle, followed by chloroform extraction and centrifugation to separate the phases. The aqueous (top) layer was collected and purified using the NEB Monarch RNA Cleanup Kit according to the manufacturer’s instructions.Sample Collection - For RNA-seq of embryos injected with water or α-amanitin, 40 embryos were flash-frozen in TRIzol immediately upon reaching nuclear cycle 14 (NC14), staged using the PCNA-GFP signal.\Library Construction - RNA-seq libraries were prepared from total RNA using the Watchmaker mRNA Enrichment and Library Prep Kits, following the manufacturer’s protocol. Libraries were dual-indexed, pooled, and sequenced.Library Construction - Transposition was carried out using 2.5 µL Tn5 transposase in a 10 µL reaction volume at 37°C for 30 minutes. Transposed DNA was purified using a MinElute cleanup kit (Qiagen) and amplified by PCR with indexed primers. Final libraries were cleaned using SPRI bead purification (1.2× ratio) and quantified by Qubit prior to sequencing.Sample Collection - For Pico-C, we adapted the fixation and sorting procedure described in (Blythe and Wieschaus, 2015) and (Ing-Simmons et al., 2022) with modifications for our low input Micro-C (Pico-C). After the initial crosslinking with formaldehyde, the reaction was quenched using 2M Tris-HCl pH 7.5 (final concentration 0.75M). The embryos were then washed with PBST, and a second crosslinking step was performed with the long crosslinker EGS (Sigma) at a final concentration of 3mM in PBST for 45 minutes at room temperature with passive mixing. The reaction was quenched again with Tris-HCl pH 7.5 (final concentration 0.75M) for 5 minutes. Afterward, the embryos were washed, sorted under a microscope, snap-frozen in liquid nitrogen, and stored at -80°C.OrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesData Transformation - Low input Micro-C (Pico-C) reads were mapped to the dm6 genome using BWA-MEM (version 0.7.17). Aligned reads were then processed with Pairtools (version 1.0.3; Abdennur et al., 2023) using the parse2 function (reporting outer position) to rescue complex walks and any reads below a mapping quality of 3 were removed. The resulting pairs files were sorted and converted to FAN-C pairs format (version 0.9.18; Kruse et al., 2020). Duplicates were removed and QC metrics were extracted. Merged FAN-C .hic files were converted to balanced cooler (version 0.8.6; Abdennur and Mirny, 2020) .mcool files using cooler zoomify with the mad-max set to 0.Data Transformation - RNA-seq libraries were aligned using STAR, and normalization was performed using size factors calculated by DESeq2. Final bigwigs were created by averaging replicates using deepTools.MetabolomicsUnknownTranscriptomicsGenomicsProteomicsIllumina NovaSeq 6000Illumina NovaSeq XNextSeq 2000Hi-CDrosophila melanogasterJuan VaquerizasYadwinder KaurNoura MaziakMelissa HarrisonHaley BrownfalseLow Input Micro-C (Pico-C) data of Drosophila melanogaster embryos across early nuclear cyclesLow-input Micro-C (Pico-C) was performed on hand-sorted PCNA-eGFP embryos at interphase from nuclear cycles (NCs) 9 to 14, including double knockdowns of Zelda and GAF and their respective controls (NC14), as well as embryos injected with α-amanitin or water (NC14). ATAC-seq was performed on double knockdowns of Zelda and GAF and their respective controls. RNA-seq was performed on α-amanitin- and water-injected embryos.2025-11-10T00:00:00Z2025-12-01T22:03:04.889Z2024-09-26T15:56:31.522ZE-MTAB-14477ERP164544EFO_0007693EFO_0002944EFO_0004170EFO_0005518EFO_0003816EFO_0004184