2025-11-04
ENA first public
ERC000011
ENA-CHECKLIST
EMBL
INSDC center name
public
INSDC status
ERS22466817
SRA accession
E-MTAB-14663:S2_SMF_DE_rep2_LSK114
Submitter Id
ArrayExpress
broker name
S2
cell_line
embryonic cell
cell_type
not collected
collection date
fruit fly
common name
Protocols: 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 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. 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.
description
not collected
geographic location (country and/or sea)
not applicable
isolate
Drosophila melanogaster
organism
Drosophila melanogaster
organism
embryonic tissue
organism part
Drosophila melanogaster
scientific_name
S2_SMF_DE_rep2_LSK114
title