GSM1656384: COL ChIP Seq; Drosophila melanogaster; ChIP-Seq
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
TFs and others
Antigen
kn
Cell type
Cell type Class
Embryo
Cell type
13-14h embryos
NA
NA
Attributes by original data submitter
Sample
source_name
whole embryos
developmental stage
stage 13-14
chip antibody
Mix of 3 anti-COL home made monoclonal antibodies
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Dounced fixed embryos where used to obtain nuclear fraction, then chromatine was sonicated. Cleared nuclear extract were used to perfomed immunoprecipitation. Competitive elution was made with a COL recombinant protein in order to release precipitated Col-DNA complexes. ChIP-seq libraries were prepared using ChIP-Seq Sample Prep Kit (#IP-102-1001, Illumina) following the manufacturer's protocol with some modifications. Briefly, 10 ng of ChIP enriched DNA or control DNA were end repaired using T4 DNA polymerase, Klenow DNA polymerase and T4 PNK. A single 'A' nucleotide was added to the 3' ends of the blunt DNA fragments with a Klenow fragment (3' to 5'exo minus). The ends of the DNA fragments were ligated to double stranded adapters using T4 DNA Ligase. The ligated products were enriched by PCR (30 sec at 98¡C; [10 sec at 98¡C, 30 sec at 65¡C, 30 sec at 72¡C] x 14 cycles; 5 min at 72¡C) and then purified using Agencourt AMPure XP beads (#A63881, Beckman). DNA library size selection was performed by excising the 250-350 bp fragments from a 2% agarose gel followed by purification using a QIAquick Gel Extraction Kit (#28906, Qiagen). Prior to analysis DNA libraries were checked for quality and quantified using a 2100 Bioanalyzer (Agilent). The libraries were loaded in the flowcell at 8pM concentration and clusters were generated using the Cbot and sequenced on the Illumina Genome Analyzer IIx as single-end 54 base reads following Illumina's instructions