GSM5660002: GFP dsRNA Ez IP Rep 2; Drosophila melanogaster; ChIP-Seq
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
TFs and others
Antigen
E(z)
Cell type
Cell type Class
Cell line
Cell type
S2
Source
Oregon R
Developmental Stage
late embryonic stage
Attributes by original data submitter
Sample
source_name
Schneider 2 (S2) cells
strain
Oregon R
developmental stage
Late embryonic stage
antibody
rabbit anti-E(z) (from Richard Jones)
spike-in
N/A
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Cells were fixed in 1% formaldehyde (Sigma Aldrich, F8775) for 15 min at room temperature, quenched with 0.16 M glycine for 5 min at 4°C and washed in cold PBS. Crude nuclear extracts were obtained by sequentially washes with ChIP buffer A (10 mM HEPES pH 7.6, 10 mM EDTA pH 8.0, 0.5mM EGTA pH 8.0 and 0.25% Triton X-100) followed by ChIP buffer B (10mM HEPES pH 7.6, 100 mM NaCl, 1 mM EDTA pH 8.0, 0.5 mM EGTA pH 8.0 and 0.01% Triton X-100) to isolate nuclei. Nuclei were resuspended in Sonication buffer (15 mM HEPES pH 7.6, 140 mM NaCl, 1 mM EDTA pH 8.0, 0.5 mM EGTA pH 8.0, 0.1% sodium deoxycholate and 1% Triton X-100), with Complete Mini EDTA-Free protease inhibitor tablets, Roche) supplemented with 0.5% SDS and 0.2% n-lauroylsarcosine, at a final concentration of 5 – 10 x 107 cells/ml and sonicated in a Bioruptor (Diagenode) using high power settings to obtain an average fragment size distribution of 200–500 bp. The concentration of chromatin was quantified. Immunoprecipitations were performed by incubating 10 μg of chromatin with anti-CBP (homemade), anti-E(z) (kind gift of Richard Jones, Southern Methodist University, USA) and anti-Pho (kind gift of Judith Kassis, National Institute of Health, USA) overnight. Chromatin-antibody complexes were bound to Protein A and G Dynabeads (Invitrogen) and washed sequentially with Wash A (10 mM Tris pH 8.0, 1 mM EDTA pH 8.0, 140 mM NaCl, 0.1% SDS, 0.1% Sodium Deoxycholate and 1% Triton X-100), Wash B (Wash A adjusted to 500 mM NaCl), Wash C (10 mM Tris pH 8.0, 1 mM EDTA pH 8.0, 250 mM LiCl, 0.5% Sodium Deoxycholate and 0.5% IGEPAL CA-630) and Tris-EDTA (TE). Beads were resuspended in 100 μl TE, treated with RNase A (20 μg/ml) at 55°C for 30 min, supplemented with SDS (0.75%) and Tris (50 mM). and crosslinks reversed at 65°C overnight. Eluted ChIP DNA was treated with Proteinase K at 55°C for 2 h and purified using the ChIP DNA Clean & Concentrator kit (ZymoResearch, D5205). Libraries were constructed from ChIP samples (2-5 ng) with the NEBNext Ultra II DNA Library Prep Kit (NEB) and single-end (1x75 bp) sequenced on an Illumina NextSeq 550 platform at the BEA core facility, Stockholm.