GSM3231637: Kelly, Input.H3K27Ac.NaiveVeh; Homo sapiens; ChIP-Seq
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
Input control
Antigen
Input control
Cell type
Cell type Class
Neural
Cell type
KELLY
Primary Tissue
Brain
Tissue Diagnosis
Neuroblastoma
Attributes by original data submitter
Sample
source_name
Kelly neuroblastoma cell line
chip antibody
Input
status
naïve
treatement
vehicle
cell line
Kelly
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Cells were harvested, washed twice in ice cold PBS with protease inhibitors and then lysed in 50 mM Tris-HCl pH 8.1, 10 mM EDTA, 1% SDS supplemented with protease inhibitors (Roche). Chromatin was sheared to ~250 bp fragments using the Covaris ultra-sonication. The sonicated lysate was then diluted with ChIP dilution buffer (16.7 mM Tris-HCl pH 8.1, 16.7 mM NaCl, 1.2 mM EDTA, 1.1% Triton-X100, 0.01% SDS). Subsequently, 2.5% of the lysate was removed as the input control. The remaining lysate was incubated over night at 4°C with 3 µg of H3K27Ac antibody (ab4729, Abcam). The precipitated lysate was then incubated with protein A/G sepharose beads (Pierce, catalog #20423) for 2 hours and then washed sequentially in ice cold low salt buffer (20 mM Tris-HCl pH 8.1, 150 mM NaCl, 2 mM EDTA, 1% Triton-X100, 0.1% SDS), high salt buffer (20 mM Tris-HCl pH 8.1, 2 mM EDTA, 500 mM NaCl, 1% Triton-X100, 0.1% SDS), LiCl buffer (10 mM Tris-HCl pH 8.1, 0.25 M LiCl, 1 mM EDTA, 1% deoxycholic acid, 1% IGEPAL CA-630) and TE buffer (10 mM Tris-HCl pH 8.1, 1 mM EDTA). Samples were incubated at 65°C overnight with elution buffer (0.1 M NaHCO3, 1% SDS) supplemented with 0.2M NaCl to reverse crosslinking. ChIP libraries were prepared using the Rubicon Genomics Thruplex DNA-seq prep kit optimized for low input samples.