progeny of mat-a-tubGal4/mofRNAi mothers crossed to their siblings (Bloomington stocks: 7063 & 58281)
tissue
preblastoderm embryos nc9-13
technique
ATAC-seq
Sequenced DNA Library
library_strategy
ATAC-seq
library_source
GENOMIC
library_selection
other
library_construction_protocol
ATAC-seq: D. melanogaster dechorionated embryos were fixed using 1% formaldehyde for 15min at RT. 25 or 100 embryos were hand staged based on morphology for cycle 9-13 or Mid nuclear cycle 14 corresponding to st3-4 and st5 respectively. Nuclei isolation was achieved using Covaris sonication and nuclei filtering. Nuclei pellet was dissolved in 50ul of cold buffer containing 10mM Tris- HCl pH=7.4, 10 mM NaCl, 3 mM MgCl2, 15 mM HEPES (pH 7.6) and 0.1% NP-40. A 10min centrifugation at 500g followed. Further processing for ATAC-seq was adapted from (31) Briefly, the nuclei pellet was resuspended in Tn5 Reaction mix containing 2.5 Tn5 from the Nextera DNA Library Prep Kit from Illumina (cat. No 15027987), 25ul Buffer 2x and 22.5ul H20. The tagmentation reaction took place at 37o shaking at 800rpm. After precisely 30min, the samples were placed on ice and 250ul of PB buffer from the MinElute PCR Purification Kit (cat. No 28004) was added to stop the reaction. DNA purification was followed according to manufacture's instructions. Pure DNA was amplified using 12 PCR cycles using the NebNext Polymerase (M0541S) for the library amplification. Nextera