Sample information curated by ChIP-Atlas

Antigen

Antigen Class
Input control
Antigen
Input control

Cell type

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

Attributes by original data submitter

Sample

source_name
LNCaP cells
passages
5
antibody
none

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Cells were then cross-linked with 1% formaldehyde for 10 min at 37 °C. Cells were then placed on ice and washed with ice cold PBS (2 x 10 mL), scraped and pelleted. Pellets were then resuspended in PIPES buffer (5 mM PIPES (pH 8.0), 85 mM KCl, 0.5% NP-40, 1x cOmplete, EDTA-free, Protease Inhibitor Cocktail Tablets (Roche)), lysed in lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl pH 8.1, 1x cOmplete, EDTA-free, Protease Inhibitor Cocktail Tablets (Roche)), and sonicated to shear cross-linked DNA. Samples were kept in an ice bath at all times. Nucleic acid concentration was then measured using a Nanodrop (Thermo Scientific). The nucleic acid (20-100 μg) was then resuspended in 450-1,000 μL 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, 1x cOmplete, EDTA-free, Protease Inhibitor Cocktail Tablets (Roche)), and pre-cleared by adding 30 μL Protein A Dynabeads (Invitrogen) and rotated for 30 minutes at 4°C. Samples were then incubated overnight at 4 °C with 5 μg of polyclonal rabbit H3K4Me2 (milipore 07-030) (a no antibody control sample was included). 65 μL Dynabeads were then added to the samples and rotated for 2 h at 4°C. Dynabeads were then washed 2x with a low salt wash (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris pH 8.1, 150 mM NaCl); 1x with LiCl wash (0.25 M LiCl, 0.5% NP-40, 0.5% Na Deoxycholate, 1 mM EDTA, 10 mM Tris-HCl pH 8.1); and 2x with TE pH 8.0. Elution buffer was then added to the beads (1% SDS, 0.1 M NaHCO3) and samples were vortexed and rotated at RT for 15 minutes and sample transferred to a new tube. This step was repeated 2x. Cross-linking was reversed by the addition of 20 μL 5 M NaCl and heating at 65°C for 4 h. 10 mM EDTA, 40 mM Tris-HCl pH 6.5, and 40 μg Proteinase K (Thermo Scientific #EO0491) were then added and samples were incubated for 1 h at 45 °C. 500 μL phenol:chloroform was then added to the samples and they were rotated overnight at 4°C. Samples were then spun and the top layer (aqueous) was placed in a new tube. An equal volume of chloroform was added and vortexed and spun and the bottom layer discarded again. 50 μg mL-1 of GlycoBlue (Life Technologies AM9515), 0.5 M NaOAc pH 5.2, and 2 volumes of 100% ethanol was added and samples were placed on ice for 15 minutes. Samples were then spun down and pellet was washed with 1 volume 70% EtOH and let dry. The pellets were then resuspended in TE and DNA concentrations were quantified by Qubit assay HS kit (Invitrogen Q32851). Libraries were prepared from 10-20 ng of IP ChIP DNA and 100 ng of input DNA according to Illumina’s instructions along with the ChIP-seq DNA Sample Prep Kit (IP-102-1001). Briefly, samples were checked for quality and concentration from 150-250 bp on a bioanalyzer. DNA was end-repaired using Klenow polymerase in 58 μL of reaction buffer. For IP DNA, Klenow was diluted 1:5. Samples were incubated at 20°C for 30 minutes and subsequently purified on QIAquick PCR purification columns. A-tails were then added to the DNA with Klenow and dATP in NEB buffer 2 at 37°C for 30 minutes and cleaned with Qiagen MiniElute PCR purification columns. Sequencing adapters were then ligated onto the DNA for 15 minutes at room temperature followed by cleaning with MiniElute columns. Samples were then run on 2% agarose gels and DNA from 216-366 bp (DNA plus adapters) were cut from the gel and purified with a Qiagen QIAquickGel Extraction kit. Concentrations were then checked on a bioanalyzer and 8 ng were PCR amplified with Phusion polymerase (Fisher) for 15 cycles (10 sec 98°C, 30 sec 65°C, 30 sec 72°C) followed by 5 minutes at 72°C. Samples were then cleaned with Ampure kits (Illumina) and washed with 80% ethanol. DNA samples were resuspended at the end of the cleanup into 17.5 μL buffer EB (Qiagen) and subjected to next generation sequencing on Illumina HiSeq platform according to manufacturers instructions.

Sequencing Platform

instrument_model
Illumina HiSeq 2000

hg38

Number of total reads
16307915
Reads aligned (%)
97.4
Duplicates removed (%)
0.1
Number of peaks
474 (qval < 1E-05)

hg19

Number of total reads
16307915
Reads aligned (%)
96.4
Duplicates removed (%)
0.1
Number of peaks
405 (qval < 1E-05)

Base call quality data from DBCLS SRA