Sample information curated by ChIP-Atlas

Antigen

Antigen Class
TFs and others
Antigen
NXF1

Cell type

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

Attributes by original data submitter

Sample

source_name
Nxf1-ChIP-1
transgenic construct
FLAG-Nxf1
antibody
FLAG
replicate
1
cell line
HEK293T

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Cell pellets were lysed in ChIP Lysis Buffer 1 (50 mM HEPES-NaOH pH7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP40, 0.25% Triton X-100, protease inhibitors SigmaFAST) and rotated at 4°C for 5 minutes. Nuclei were then pelleted via centrifugation (3000 x g, 5 minutes at 4°C), and resuspended in ChIP Buffer 2 (10 mM Tris-HCl pH 7.3, 200 mM NaCl, 0.5 mM EGTA, 1 mM EDTA, protease inhibitors SigmaFAST), before rotation at room temperature for 10 mins. Nuclei were pelleted via centrifugation (1500 x g, 5 minutes at 4°C) and resuspended in ChIP Lysis Buffer 3 (10 mM Tris-HCl pH 7.3, 200 mM NaCl, 0.5 mM EGTA, 1 mM EDTA, 0.1% Na-deoxycholate, 0.5% N-lauroylsarcosine, protease inhibitors SigmaFAST). Samples were sonicated using a Bioruptor (High, 20 x [30s-ON/30s-OFF]) to generate chromatin fragments of 250-300 nts, and cleared by centrifugation (16100 x g, 15 minutes, 4°C). Lysate concentrations were measured by Bradford assay, and equal concentrations of chromatin were incorporated into the IPs. IPs were carried out overnight at 4°C using 5 µg of FLAG antibody (Sigma). 100 µL blocked protein-G Dynabeads were then added to the samples and incubated for 2 hours at 4°C. Following incubation, beads were washed with 4 x 0.5 mL RIPA Wash Buffer (50 mM HEPES-NaOH pH 7.5, 500 mM LiCl, 1 mM EDTA, 1% NP40, 0.7% Na-deoxycholate, 0.1% N-lauroylsarcosine) and once with 0.5 mL ChIP Final Wash Buffer (10 mM Tris-HCl pH7.3, 1 mM EDTA, 50 mM NaCl). Complexes were eluted by adding 200 µL of ChIP Elution buffer (50 mM Tris-HCl pH8.0, 10 mM EDTA, 1% SDS) and incubated at 65°C for 30 minutes. NaCl was added to a final concentration of 200 mM and cross-links were reversed overnight at 65°C. Samples were then treated with RNase A (0.2 mg/mL) for 2 hours at 37°C, followed by proteinase K (0.2 mg/mL) for 2 hours at 55°C. DNA was purified via phenol-chloroform extraction and ethanol precipitation, and then resuspended in water. Library construction was using the standard ChIP-seq protocol of Novogene (Beijing, China)

Sequencing Platform

instrument_model
Illumina NovaSeq 6000

hg19

Number of total reads
24475173
Reads aligned (%)
167.6
Duplicates removed (%)
9.8
Number of peaks
434 (qval < 1E-05)

hg38

Number of total reads
24475173
Reads aligned (%)
169.3
Duplicates removed (%)
9.5
Number of peaks
791 (qval < 1E-05)

Base call quality data from DBCLS SRA