GSM3068975: HEK Ub-E1 HS RPB1 ChIP Rep2; Homo sapiens; ChIP-Seq
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
RNA polymerase
Antigen
RNA polymerase II
Cell type
Cell type Class
Kidney
Cell type
293
Primary Tissue
Kidney
Tissue Diagnosis
Normal
Attributes by original data submitter
Sample
source_name
Flp-In T-REx 293 Cell Line
chip antibody
RNA PolII (RPB1) Cell Signaling 14958
treatment
NSC624206 (Ub-E1 inhibitor)
condition
Heat-Shock
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Cells were fixed using 1% methanol-free formaldehyde (CAT no) in DMEM (HEK293) or IMDM (K562) at room temperature for 10 minutes, followed by 5 minutes blocking in 0.125M glycine. Cells were washed twice with ice-cold PBS. Cell pellet was resuspended in Farnham Buffer (5 mM PIPES pH 8; 85 mM KCl; 0.5% Igepal). Cell suspensions were sonicated in 1ml Covaris tubes using Covaris S220 with the following settings: Peak Power=75; Duty Factor=2; Cycles/Burst=200. Sonication time varied from cell type to cell type; K562=~2.5minutes and HEK293=~3minutes. Isolated nuclei were washed with Farnham Buffer and suspended in shearing buffer (10 mM Tris-HCl pH 8; 0.1% SDS; 1 mM EDTA). Chromatin was sheared by sonication in 1ml Covaris tubes using the following settings: Peak Power=140; Duty Factor=5; Cycles/Burst=200, Time=25-30 minutes. Debris was removed by centrifugation. A DNA fragment-size distribution of 200-600bp was considered as ideal chromatin for ChIP. Chromatin was diluted 1:1 with IP buffer to achieve a final 0.05% SDS concentration. 200 μg of good quality chromatin was used for immunoprecipitation. Protein A or G magnetic beads (CAT) were incubated (rotated) with 5-10μg of antibody for 6h at 4ºC. This bead-antibody complex was then incubated overnight at 4ºC with chromatin.