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
ERS25212028
SRA accession
E-MTAB-14477_2:water_rnaseq2
Submitter Id
1-2.5
hour
age
ArrayExpress
broker name
not collected
collection date
fruit fly
common name
Protocols: For RNA-seq of embryos injected with water or α-amanitin, 40 embryos were flash-frozen in TRIzol immediately upon reaching nuclear cycle 14 (NC14), staged using the PCNA-GFP signal.\" Pico-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% monomer vs ~30% dimer. Size selection was not carried out via gel-extraction but done with AMPure XP (Beckman Coulter) beads after library amplification.  For library construction, samples were homogenized using a metal pestle, followed by chloroform extraction and centrifugation to separate the phases. The aqueous (top) layer was collected and purified using the NEB Monarch RNA Cleanup Kit according to the manufacturer’s instructions. RNA-seq libraries were prepared from total RNA using the Watchmaker mRNA Enrichment and Library Prep Kits, following the manufacturer’s protocol. Libraries were dual-indexed, pooled, and sequenced.
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
water_rnaseq2
title