title: GSM8804949: Embryo_GFPCBP_Zld_Input1; Drosophila melanogaster; ChIP-Seq
library_construction_protocol: The ChIP protocol used on fixed embryos in this study was adapted from a published protocol (Blythe and Wieschaus, 2015). Embryos were collected 2-3hrs AEL, dechorionated in 50% bleach for 3 min, and followed by a 15min fixation in .45% formaldehyde for Zld. Two replicates of 1000 stage five embryos per experiment were hand sorted under a dissecting scope to avoid contamination by older embryos. Sorted embryos were homogenized in 1mL of RIPA buffer (50 mM Tris-HCl pH 8.0, 0.1% SDS, 1% Triton X-100, 0.5% sodium deoxycholate, and 150 mM NaCl) and underwent 11 cycles of sonication for 20s at 20% output on full duty cycle. For Zld ChIP in zld-RNAi, a spike-in of fixed and sonicated D. virilis was added before removing 2% input. IPs were incubated with 7 μl antibody overnight at 4C and purified using Protein A magnetic beads (Invitrogen). After washing and eluting purified IP, DNA from IPs and inputs were treated with 90µg of RNAseA for 30mins at 37˚C and decrosslinked by adding 100µg of Proteinase K overnight at 65˚C. DNA was purified using a phenol:chloroform extraction with an overnight ethanol precipitation step to concentrate samples. Libraries for inputs and IPs were prepared using the commercial NEB Next Ultra II kit. Libraries were contructed using the NEB Next II Ultra kit. Sequencing was completed on the Illumina NovaSeq X Plus using 150bp pair end reads.
identifiers.namespace: GEO
identifiers.id: GSM8804949_r1