Sample information curated by ChIP-Atlas

Antigen

Antigen Class
TFs and others
Antigen
FANCM

Cell type

Cell type Class
Uterus
Cell type
HeLa
Primary Tissue
Cervix
Tissue Diagnosis
Adenocarcinoma

Attributes by original data submitter

Sample

source_name
FANCM ChIP-Seq
cell line
Hela/GFP-DONSON cells
chip antibody
Bethyl A302-637A, Rabbit anti FANCM

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Cells were crosslinked with 1% formaldehyde in culture media for 8 min, followed by quenching the formaldehyde with 0.1 M glycine. Cells were washed with PBS, harvested by scraping, then suspended in lysis buffer (0.5% SDS, 10 mM EDTA, 50 mM Tris-HCL pH 8.0) supplemented with protease and phosphatase inhibitors. Lysates were sonicated in a 4 ⁰C water bath ultrasonicator (Bioruptor, Diagenode). The time of sonication was adjusted to yield short DNA fragments <500 bp (total 8 minutes, 30 seconds sonication, then cool 30 seconds). In some experiments the time was adjusted to yield longer DNA fragments of 500-5000 bp (2 x 30 seconds with a 30 second cooling period). Diluted lysates were incubated overnight at 4 °C with antibodies as indicated. Immunoprecipitations were performed using Protein G magnetic beads (Pierce, 10% v/v), or GFP Trap (Chromotek, gta-20). Bead bound complexes were washed with low salt immune complex buffer (0.1% SDS, 1% Triton x-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 150 mM NaCl), high salt immune complex buffer (0.1% SDS, 1% Triton x-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 500 mM NaCl), LiCl immune complex buffer (0.25 M LiCl, 1% NP-40, 1% mM EDTA, 10 mM Tris-HCl pH 8.0) and TE buffer (10 mM Tris-HCl, 1 mM EDTA pH 8.0). DNA was eluted in elution buffer (1% SDS, 0.2 M NaCl) with Proteinase K (100 µg/ml) overnight at 65 °C. Eluted DNA was purified with DNA Clean & Concentrator PCR purification Kit (ZYMO Research, D4033) according to the manufacturer instructions. Illumina sequencing adapters with a T-overhang were ligated to the precipitated ChIP DNA fragments or the input DNA, with a corresponding A-overhang, to construct a sequencing library according to the manufacturer's protocol (Illumina, San Diego, CA). The fragments were purified using a magnetic bead protocol and eighteen cycles of PCR amplification were performed to enrich for fragments with an adapter on both ends. The products were purified again with size selection (approximately 200–600 bases) using a dual bead selection protocol with SPRIselect Beads (Beckman Coulter, Brea, CA). These libraries were sequenced on an Illumina Hi-Seq 2500 sequencer using on-board cluster generation on a rapid run paired end flow cell for 75 X 75 cycles (DONSON) and single end of 75 bp for 75 cycles (FANCM). Real-time analysis was performed using RTA v1.18.66.3 and base-calling was performed using bcl2fastq v2.18.0.12.

Sequencing Platform

instrument_model
Illumina HiSeq 2500

hg19

Number of total reads
167960519
Reads aligned (%)
81.6
Duplicates removed (%)
15.5
Number of peaks
2491 (qval < 1E-05)

hg38

Number of total reads
167960519
Reads aligned (%)
84.1
Duplicates removed (%)
13.6
Number of peaks
3713 (qval < 1E-05)

Base call quality data from DBCLS SRA