2025-09-16
ENA first public
ERC000011
ENA-CHECKLIST
MRC Laboratory of Medical Sciences, London, UK Institute of Clinical Sciences, Faculty of Medicine, Imperial College London, London, UK
INSDC center name
public
INSDC status
ERS21097824
SRA accession
E-MTAB-14477:NC14_rep6
Submitter Id
1-2.5
hour
age
ArrayExpress
broker name
not collected
collection date
fruit fly
common name
Protocols: 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. 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 ~70-80% monomer vs 30-20% dimer. Size selection of di-nucleosomal DNA was not carried out via gel-extraction but done with AMPure XP (Beckman Coulter) beads after library amplification.  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.
description
NC14
developmental stage
yw; eGFP-PCNA
genotype
not collected
geographic location (country and/or sea)
not applicable
isolate
Drosophila melanogaster
organism
Drosophila melanogaster
organism
whole embryo
organism part
Drosophila melanogaster
scientific_name
mixed
sex
yw; eGFP-PCNA
strain
NC14_rep6
title