biostudies-arrayexpresscharles girardotDrosophila melanogasterhttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-17018Collection of 2 biological replicates of SMF-ONT performed in S2 cell incubated for 72hrs with RNAi targeting Clamp or LacZ (ctrl) transcripts. 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 (LSK114) preparation has been performed using 1µg of purified footprinted HWM DNA. Sequencing has been performed using 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 - Sequencing has been performed using promethION flow cells for the R10 chemistry (LSK114)Sample Treatment - 10 μg of each purified dsRNA was mixed for each target to a total of 30 μg and directly added to 1.5x10^6 of S2 cells in a 6 well plate and incubated for 72 hr at 25 °C.Library 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 30°C. CpG methylation is performed adding 60U of SssI, 10mM MgCl2, and 0.38mM SAM and incubating for 7.5 minutes at 30°C. Enzymes are inactivated at 65°C for 20 minutes, and 400µg of proteinase K is added, with the reaction incubating for over 5 hours at 55°C. Finally, the footprinted DNA is purified using the Quick-DNA[TM] HMW MagBead Kit (Zymo) as per the manufacturer's instructions.Sample Collection - D. melanogaster S2 cells are cultured at 25°C 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 FilesData Transformation - The data have been analysed using the SMF-ONT nextflow pipeline (https://git.embl.de/grp-krebs/nf-smfont) using custom methylation call models.MetabolomicsUnknownTranscriptomicsGenomicsProteomicsPromethIONRNA-seq of coding RNADrosophila melanogasterYuliya Badayevacharles girardotMathias BoulangerArnaud KrebsChatsirisupachai KasitfalseSMF-ONT S2 clamp RNAi knockdownCollection of 2 biological replicates of SMF-ONT performed in S2 cell incubated for 72hrs with RNAi targeting Clamp or LacZ (ctrl) transcripts. 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 (LSK114) preparation has been performed using 1µg of purified footprinted HWM DNA. Sequencing has been performed using 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.2026-08-04T00:00:00Z2026-08-04T01:00:48.235Z2026-05-06T12:48:50.251ZE-MTAB-17018ERP193084EFO_0002944EFO_0004170EFO_0005518EFO_0003816EFO_0003738EFO_0004184EFO_0003969