SMF-ONT in S2 and OSC Drosophila cell lines
SMF-ONT in S2 and OSC Drosophila cell lines
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. 2 biological replicates of SMF-ONT performed in S2 and OSC cell lines 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 Âµg 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. 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.