biostudies-arrayexpressMetabolomicsUnknownTranscriptomicsGenomicsProteomicsCharles GirardotGridIONPromethIONDNA-seqDrosophila melanogasterDrosophila melanogasterhttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-14663Collection of 2 biological replicates of SMF-ONT performed in S2 and OSC cell lines. The footprinting protocol was adapted from Kleinendorst and Barzaghi et al., 2021 and optimised for long-read sequencing and high weight DNA extraction. In summary, 2.5 million intact nuclei per SMF reaction have been treated with successive incubation with GpC (M.CviPI) and CpG (M.SssI) methyltransferase enzymes. Footprinted DNA was extracted using the Quick-DNA HMW MagBead kit following the manufacturer protocol and using wide-bore tips. Nanopore ligation library (LSK109 or LSK114) preparation has been performed using 1µg of purified footprinted HWM DNA. Sequencing has been performed using GridION flow cells for the R9 chemistry (LSK109) and promethION flow cells for the R10 chemistry (LSK114). The data have been analysed using the SMF-ONT nextflow pipeline (https://git.embl.de/grp-krebs/nf-smfont) using custom methylation call models.biostudies-arrayexpressSequencing - single-end sequencing on oxford nanopore PromethION P2Sequencing - single-end sequencing on oxford nanopore PromethIONGrowth Protocol - Drosophila ovarian OSC cells have been cultured at 25C (without CO2) in Cross and Sang M3 medium (Shields and Sang M3 Insect Medium supplemented with 0.6 mg/ml glutathione, 10 mU/ml insulin, 10% inactivated fetal bovine serum and 5% fly extract 20X from VDRC). For maintenance, cells were split once/twice a week diluted 1:5 for no more than 20 passages. For splitting, OSCs were washed in PBS and trypsinized (with trypsin-EDTA) for 2min at 25C.Library Construction - Library preparation was performed using the ligation sequencing kit SQK-LSK109 following the manufacturer protocol. 50 fmol of DNA were loaded in the R9.4.1 flow cell.Sample Collection - 2 biological replicates of SMF-ONT performed in S2 and OSC cell linesLibrary Construction - Library preparation was performed using the ligation sequencing kit SQK-LSK114 following the manufacturer protocol. 20 fmol of DNA were loaded in the R10.4.1 flow cell.Nucleic Acid Extraction - This protocol outlines a Single Molecule Footprinting technique optimized for long-read sequencing in Drosophila cell lines, based on protocols from the Krebs lab (Barzaghi et al., 2021) and the SMAC-seq method (Shipony et al., 2020). Cells are collected, washed with cold PBS, and lysed on ice for 10 minutes using a lysis buffer (10mM Tris pH 7.4, 10mM NaCl, 3mM MgCl2, 0.1mM EDTA, 0.5% NP-40). Following lysis, wide-bore tips are used exclusively. Nuclei are isolated by centrifugation at 1000g and 4C for 5 minutes, washed with a similar buffer without detergent (10mM Tris pH 7.4, 10mM NaCl, 3mM MgCl2, 0.1mM EDTA), and centrifuged again. The nuclei are then resuspended in 1X M.GpC buffer with 0.96mM SAM and 450mM sucrose. An initial 200U of M.CviPI is added and incubated for 7.5 minutes at 30C, followed by an additional 100U of M.CviPI and 0.38mM SAM for another 7.5 minutes at 30C. CpG methylation is performed adding 60U of SssI, 10mM MgCl2, and 0.38mM SAM and incubating for 7.5 minutes at 30C. Enzymes are inactivated at 65C for 20 minutes, and 400 µg of proteinase K is added, with the reaction incubating for over 5 hours at 55C. Finally, the footprinted DNA is purified using the Quick-DNA[TM] HMW MagBead Kit (Zymo) as per the manufacturer's instructions.Sequencing - single-end sequencing on oxford nanopore GridIONGrowth Protocol - D. melanogaster S2 cells are cultured at 25C in Schneider's Drosophila medium (Gibco) supplemented with 10% of inactivated fetal bovine serum. For maintenance, cells were split every 3 to 5 days at ~1-2 million cells per mL for no more than 20 passages.OrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesCharles GirardotMathias BoulangerArnaud KrebsfalseSMF-ONT in S2 and OSC Drosophila cell linesCollection of 2 biological replicates of SMF-ONT performed in S2 and OSC cell lines. The footprinting protocol was adapted from Kleinendorst and Barzaghi et al., 2021 and optimised for long-read sequencing and high weight DNA extraction. In summary, 2.5 million intact nuclei per SMF reaction have been treated with successive incubation with GpC (M.CviPI) and CpG (M.SssI) methyltransferase enzymes. Footprinted DNA was extracted using the Quick-DNA HMW MagBead kit following the manufacturer protocol and using wide-bore tips. Nanopore ligation library (LSK109 or LSK114) preparation has been performed using 1µg of purified footprinted HWM DNA. Sequencing has been performed using GridION flow cells for the R9 chemistry (LSK109) and promethION flow cells for the R10 chemistry (LSK114). The data have been analysed using the SMF-ONT nextflow pipeline (https://git.embl.de/grp-krebs/nf-smfont) using custom methylation call models.2026-08-04T00:00:00Z2026-08-04T01:00:48.237Z2024-11-29T20:30:25.85ZE-MTAB-14663EFO_0002944EFO_0004170EFO_0002693EFO_0005518EFO_0004184