Sample information curated by ChIP-Atlas

Antigen

Antigen Class
Input control
Antigen
Input control

Cell type

Cell type Class
Kidney
Cell type
293
Primary Tissue
Kidney
Tissue Diagnosis
Normal

Attributes by original data submitter

Sample

source_name
Total input Chromatin from Hek cells transfected with ZfSp5_dDBD expression plasmid
cell line
HEK293
transfected with
zFSp5_dDBD
age post-transfection
24h
antibody
anti-HA (Novus biologicals NB600-363)

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
24 hours after transfection, cells were collected by scraping, washed twice in pre-warmed culture medium and fixed in 1% FA solution (Sigma) for 15 minutes. The fixative was quenched by adding Glycine (125 mM) and incubation for 3 minutes. The cells were washed once in ice-cold PBS and re-suspended in 5 mL chromatin prep buffer (Active Motif), containing 0.1mM PMSF and 0.1% protease inhibitor cocktail (PIC). To release the nuclei the sample was transferred into a pre-cooled 15 mL glas Douncer and dounced with 30 strokes. After 10 minutes incubation on ice and centrifugation at 1250 g for 5 minutes at 4°C, the nuclei were re-suspended in 500 uL sonication buffer (1% SDS, 50 mM Tris-HCl pH 8.0, 10 mM EDTA pH 8.0, 1 mM PMSF, 1% PIC). Following another 10 minutes incubation on ice, the chromatin was sonicated with a Bioblock Scientific VibraCell 75042 sonicator (Amplitude: 25%, Time: 12 minutes, 30 seconds on, 30 seconds off, 24 cycles). Note: The sonication conditions were optimized to have a fragmentation size of around 250 bp. Then 100 uL of the sonicated chromatin was added to 900 uL ChIP dilution buffer (0.1% NP-40, 0.02 M HEPES pH 7.3, 1 mM EDTA pH 8.0, 0.15 M NaCl, 1 mM PMSF, 1% PIC) and incubated with 4 ug anti-HA antibody (NB600-363, Novus Biologicals) over night at 4°C on a rotator. Next, the sample was loaded on a ChIP-IT ProteinG Agarose Column (Active Motif) and incubated for 3 hours at 4°C on a rotator. The column was washed 6 times with 1 mL buffer AM1 (Active Motif) and the DNA eluted with 180 uL of pre-warmed buffer AM4 (Active Motif). The sample was decrosslinked by adding 30 uL 10x TE buffer, 18 uL 5 M NaCl, 57 uL H2O and incubated for 5 hours at 65°C. 5 uL of RNAse A (10 ug/uL) was added and the sample incubated at 37°C for 30 minutes before adding 10 uL of Proteinase K (10 ug/uL), and further incubation for 2 hours at 55°C. The DNA was purified with the MiniElute PCR purification kit (Qiagen). For preparing the Input DNA, 5 uL sonicated chromatin was added to 5 uL 5M NaCL in 40 uL H2O, and incubated for 15 minutes at 95°C. Next the sample was incubated at 37°C for 5 minutes with 2.5 uL of RNAse A (10 ug/ul), 2.5 uL PK (10 ug/uL) was then added, and the incubation continued at 55°C for 30 minutes. 10 uL were taken for purification (MiniElute PCR purification kit from Qiagen). 5-10 ng of purified DNA were used to make libraries according to the manufacturer's protocol (Illumina). The material was sequenced with 50 bp single-end reads on the Illumine HiSeq according to the manufacturer's specifications.

Sequencing Platform

instrument_model
Illumina HiSeq 4000

hg38

Number of total reads
85780466
Reads aligned (%)
94.5
Duplicates removed (%)
12.4
Number of peaks
9564 (qval < 1E-05)

hg19

Number of total reads
85780466
Reads aligned (%)
93.0
Duplicates removed (%)
16.4
Number of peaks
9959 (qval < 1E-05)

Base call quality data from DBCLS SRA