GSM3090182: No Antibody ChIP-seq Replicate 2; Drosophila melanogaster; ChIP-Seq
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
Input control
Antigen
Input control
Cell type
Cell type Class
Larvae
Cell type
Eye-antennal discs
NA
NA
Attributes by original data submitter
Sample
source_name
L3 eye antennal discs
developmental stage
Late L3
tissue
eye antennal discs
strain
Canton S
antibody
none
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
400 Canton S third instar larval eye-antenna discs were dissected and crosslinked with 1% formaldehyde for 15 min and quenched by 125 mM Glycine for 5 min at room temperature. After washing with PBS, discs were lysed in lysis buffer (50 mM K-Hepes pH 7.8, 140 mM NaCl, 1 mM EGTA, 1 mM EDTA, 1% Triton X-100, 0.1% Na-deoxycholate, protease inhibitors) and sonicated with a Branson sonifier to shear chromatin to ~200 - 400 bp. The chromatin lysate was precleared with protein A sepharose (GE healthcare, #17-5280-01) and incubated with antibody at 4°C overnight. Protein A sepharose was added to the lysate for 3 hours to capture chromatin complexes. After three washes with lysis buffer, once with high salt wash buffer (50 mM K-Hepes pH 7.8, 500 mM NaCl, 1 mM EGTA, 1 mM EDTA, 1% Triton X-100, 0.1% Na-deoxycholate, protease inhibitors) and once with TE (10 mM Tris pH8.0, 1 mm EDTA), chromatin complexes were eluted with 1% SDS, 10 mM Tris pH 8.0, and 1 mM EDTA at 65°C for 10 min, followed by reverse-crosslinking at 65°C overnight. ChIP samples were purified using Qiagen PCR purification kit and sequenced based on the manufacturer's instructions (Illumina, San Diego, CA)