Sample information curated by ChIP-Atlas

Antigen

Antigen Class
TFs and others
Antigen
SF3B1

Cell type

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

Attributes by original data submitter

Sample

source_name
HeLa Cell line, G1/S synchronized
cell cycle
G1/S synchronized
chip antibody
MBL D221-3 (Abcam)
cell line
HeLa

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
For ChIP-Seq, Cells were lysed using nucleosome lysis buffer (60 mM KCl, 15 mM NaCl, 5 mM MgCl2, 0.1 mM EGTA, 15 mM Tris-HCl pH 7.5, 0.2% NP-40) supplemented with 0.1 mM DTT and PPI. Lysates were then passed through a sucrose cushion (50 mM Tris-HCl pH 7.5, 5 mM MgCl2, 25 mM KCl, 1.2M Sucrose) by centrifugation at 3.5g for 15 minutes at 4 ˚C. The pellet (containing the nuclei) was resuspended in the digestion buffer (50 mM Tris-HCl pH 7.5, 4mM MgCl2, 1mM CaCl2, 0.32 M Sucrose) with 200U micrococcal nuclease (Mnase) (Worthington Biochemical) and incubated for 10 minutes at 37 ˚C. Digestion was stopped by the addition of 1 mM each of EDTA and EGTA. Nuclei were pelleted by centrifugation at 3.5g for 10 minutes at 4 ˚C. Pellet was resuspended in 500 µl solubilization buffer ( 50mM HEPES pH 7.6, 500mM LiCl, 1mM EDTA, 0.7% sodium deoxycholate, 0.1% SDS and 1% NP-40) and rotated at 4 ˚C for 1 hour followed by centrifugation at 12,000g for 10 minutes at 4 ˚C. The pellet was discarded and the supernatant, the nucleosome enriched fraction, was used for immunoprecipitations and immunoblot assays. DNA was extracted from a small aliquot of the nucleosome-enriched fraction using phenol: chloroform: isoamyl alcohol (Thermo Fisher Scientific 15593031) and run on a 2% agarose gel to confirm the expected size of the digested DNA (147 bp and multiples thereof). For immunoprecipitations, nucleosome enriched fractions were diluted to 1 ml in dilution buffer (0.005% SDS, 0.1% Triton X-100, 1.2 mM EDTA, 1.67 mM Tris HCl pH8.0, 167 mM NaCl ) and incubated with antibodies (2 µg/sample) conjugated to Sepharose A or G beads with rotation at 4 ˚C overnight. Beads were washed with dilution buffer 3x and immunoblots were performed as described above. RNAse-A treatment was performed as described in [REF Ast Paper]. ChIP-Seq Libraries for sequencing were prepared using NEBNext Ultra DNA Library Preparation Kit (NEB E7370).

Sequencing Platform

instrument_model
NextSeq 500

hg19

Number of total reads
73887835
Reads aligned (%)
88.2
Duplicates removed (%)
11.2
Number of peaks
1002 (qval < 1E-05)

hg38

Number of total reads
73887835
Reads aligned (%)
89.9
Duplicates removed (%)
9.8
Number of peaks
1101 (qval < 1E-05)

Base call quality data from DBCLS SRA