Sample information curated by ChIP-Atlas

Antigen

Antigen Class
TFs and others
Antigen
EZH2

Cell type

Cell type Class
Prostate
Cell type
LNCAP
Primary Tissue
Prostate
Tissue Diagnosis
Carcinoma

Attributes by original data submitter

Sample

source_name
LNCaP_AI_EZH2_ChIP
cell line
LNCaP
cell type
prostate cancer cells
growth type
androgen-independent growth

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Androgen-dependent prostate cancer and its androgen-independent derivative cell lines was used to do the ChIP-Seq library construction. Approximately 1 × 107 cells were fixed with 1% formaldehyd, and were resuspended in 1 mL SDS Lysis buffer (1% SDS, 10mM EDTA, 50mM Tris-HCl pH 8.1). DNA was sonicated to 200 – 500 b. After centrifugation, we took 60ul supernatant and diluted tenfold with ChIP Dilution Buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl, pH 8.1, 167 mM NaCl). The lysate was then incubated with anti-EZH2 antibody(source: Millipore, cat# 17-662, lot# 2366583) or anti-H3K27me3 antibody(source: Millipore,cat#07-449,lot# 2382150) at 4 ◦ C overnight. The immunuprecipitates were further incubated with Pierce™ Protein A/G Magnetic Beads (Thermo, 88803) for 2 hours at 4 oC. After applying to magnet and removing the supernatants, we washed beads once with Low Salt Immune Complex wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1, 150 mM NaCl) and added 1 ml of buffer to each tube, rotated at 4 ℃ for 5 min; we then placed the tube on the DynaMag-2 magnet for 30 s, then discarded supernatant. We repeated washing first with High Salt Immune Complex wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1, 500 mM NaCl) and LiCl Immune Complex wash buffer(0.25 M LiCl, 1% IGEPAL-CA630, 1% deoxycholic acid (sodium salt), 1 mM EDTA, 10 mM Tris, pH 8.1) and second with TE Buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) for a total of 5 washes. The immunoprecipitates were eluted from the beads with elution buffer (50 nM Tris 8.0, 10 mM EDTA and 1% SDS) and incubated at 65 oC overnight. After sequential RNase A and proteinase K treatment, DNA fragments were purified by phenol extraction and ethanol precipitation. ChIP-Seq was performed by using ThruPLEX® DNA-seq kit (R400427; Rubicon Genomics) according to manufacturer’s instructions. The PCR products corresponding to 300-500 bps were gel purified, quantified and stored at -80 oC until used for sequencing. ChIP-Seq sequencing was performed on NextSeq500 platform with 151 bp paired-end sequencing.

Sequencing Platform

instrument_model
NextSeq 500

hg19

Number of total reads
19193558
Reads aligned (%)
163.3
Duplicates removed (%)
1.9
Number of peaks
379 (qval < 1E-05)

hg38

Number of total reads
19193558
Reads aligned (%)
165.8
Duplicates removed (%)
1.7
Number of peaks
717 (qval < 1E-05)

Base call quality data from DBCLS SRA