GSM2746625: 786 control INPUT Histone ChIPSeq; Homo sapiens; ChIP-Seq
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
Input control
Antigen
Input control
Cell type
Cell type Class
Kidney
Cell type
786-O
Primary Tissue
Kidney
Tissue Diagnosis
Adenocarcinoma
Attributes by original data submitter
Sample
source_name
clear cell renal cell carcinoma cell line
cell line
clear cell renal cell carcinoma cell line 786-O
shRNA
off-target control
chip antibody
none
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Briefly, 20-50x10^6 cells were swelled and lysed using 0.1% IGEPAL, nuclei were collected by centrifugation through a sucrose gradient, and micrococcal nuclease digestion was optimized for each cell collection to primarily generate mononucleosomes, without overdigesting. For the ChIP step, 3 μg of each histone mark antibody was used, and 50 μg of chromatin was used for methyl marks, and 100 μg was used for acetyl marks. ChIP-seq libraries were made following well-established protocols. Briefly, 2-10 ng of DNA was end-repaired, an A-overhang was added, Illumina Truseq adapters were ligated, DNA was run on an agarose gel and size-selected using the Qiagen Gel Extraction Kit from between 300-400 bp to exclude polynucleosomes, which produced amplified libraries with sharp peaks of ~280 bp, as seen on the Bioanalyzer. Finally, libraries were PCR amplified for 10-14 cycles using KAPA Biosystems HiFi PCR Master Mix. Where not indicated, all library preparation steps involved New England Biolabs enzymes. 75 bp single-end sequencing was performed on an Illumina NextSeq 500 machine.