Sample information curated by ChIP-Atlas

Antigen

Antigen Class
TFs and others
Antigen
VDR

Cell type

Cell type Class
Prostate
Cell type
PrEC
Tissue
prostate
Lineage
epithelial
Description
prostate epithelial cell line

Attributes by original data submitter

Sample

source_name
Primary prostate epithelial cells
tissue
Primary prostate epithelial cells
treatment group
vitamin D
chip antibody
VDR

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Cross-linked using 1% formaldehyde for 10 min at RT, then 125 mM glycine for 5 min. Plates were washed 2X with ice-cold PBS, incubated in TrypLE at 37˚C for 3 min, and washed 2X with ice-cold PBS. Cells were lysed in Nuclear Isolation Buffer from Magna ChIP HiSens Chromatin Immunoprecipitation Kit (Millipore) on ice for 20 min with vortexing for 10 s every 5 min. Nuclei were pelleted at 800xg 5 min, then re-suspended in 1 ml RIPA buffer with Protease Inhibitor Cocktail III (Millipore). Cells were sonicated on ice for 15 s 9X setting 2 on a Sonic Dismembrator model 100 (Thermo Fisher). 10 µl of sonicated DNA (~10 ng) was saved as input control. 1ml of sonicated DNA (~1 µg) was used for VDR and normal IgG ChIPs. 300 µl Dynabeads (Thermo Fisher) were washed twice with 1.5 mL 1 mg/ml BSA/PBS 2 mM EDTA solution and once with 1.5 ml 5 mg/ml BSA/PBS. Beads were re-suspended in 1000 µl with 1 mg/ml BSA/PBS 2 mM EDTA solution and incubated with 24 µg of anti-VDR C-20x sc-1008x rabbit polyclonal (Santa Cruz) for 5 hrs, then washed again as above. Antibody-conjugated beads were incubated with sonicated DNA overnight rotating at 4˚C. Beads were washed 1x with 5:1 RIPA:PBS containing 5mg/mL BSA (fraction V) and Protease Inhibitor Cocktail III (Millipore), then 5x in LiCl IP wash buffer (100 mM Tris-HCl pH 7.5/ 500 mM LiCl/1% NP-40 /1% Sodium deoxycholate) and 1x in TE buffer. Bound DNA was eluted off the beads twice, with 100 µl ChIP Elution Buffer (Millipore) containing 283 mM NaCl and 1 µl proteinase K and heated at 65˚C for 1 hr with vortexing every 15 min. Eluted samples were combined and incubated at 65˚C overnight. Input 10 µl sample was combined with 90 µl ChIP Elution Buffer (Millipore) and 283 mM NaCl and 1 µl proteinase K and incubated at 65˚C along with the other samples. DNA was purified using the Clontech NucleoSpin kit (Clontech) and eluted twice using 20 µl of 70˚C elution buffer. Libraries were prepared from 10 ng of ChIP DNA with the Library Construction Kit Kapa Biosystems (Wilmington, MA) with two modifications: adaptors were diluted 1:20 and adaptor-modifed DNA were amplified for 10 cycles. The pooled libraries were sequenced on one lane each for 101 cycles on a HiSeq2500 in High Output mode using a TruSeq SBS sequencing kit version 3.

Sequencing Platform

instrument_model
Illumina HiSeq 2500

hg19

Number of total reads
45620394
Reads aligned (%)
95.1
Duplicates removed (%)
3.9
Number of peaks
3659 (qval < 1E-05)

hg38

Number of total reads
45620394
Reads aligned (%)
96.9
Duplicates removed (%)
2.6
Number of peaks
3939 (qval < 1E-05)

Base call quality data from DBCLS SRA