Sample information curated by ChIP-Atlas

Antigen

Antigen Class
Input control
Antigen
Input control

Cell type

Cell type Class
Pluripotent stem cell
Cell type
ES cells
NA
NA

Attributes by original data submitter

Sample

source_name
Female GLI2-3xFLAG Day 0 Input rep1
antibody
Anti-FLAG
cell line
F1-2.1
Sex
female
transgene
GLI2-3xFLAG

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Frozen cell pellets were resuspended in Lysis buffer 1 (50mM Tris-HCl pH 8.0, 10mM EDTA, 0.5% SDS). Chromatin was sheared in a Qsonica Q800R for 2 minutes at 40% power 30sec on 30sec off at 4C to ~500bp. 4 volumes of Lysis buffer 2 (50mM Tris-HCl pH 7.5, 187.5mM NaCl, 0.625% Triton X-100, 0.625% sodium deoxycholate, EDTA-free cOmplete Protease Inhibitor (Roche)) were added with 0.5mg RNaseA. Chromatin samples were incubated at 37C for 30minutes. Sepharose 4B (Sigma-Aldrich) was washed with ChIP Buffer (50mM Tris-HCl pH 7.6, 150mM NaCl, 0.5% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 2mM EDTA) with protease inhibitors and used to preclear RNase A treated chromatin samples. 20uL/IP anti-FLAG M2 agarose (Sigma-Aldrich) was blocked in ChIP buffer, 100ug/mL Acyclovir, 100ug/mL Zalcitabine, 5% Gly-Gly (Sigma-Aldrich), and protease inhibitors. Precleared chromatin and blocked anti-FLAG agarose were incubated together overnight at 4C with rotation. 10% of the precleared chromatin input was saved. IP samples were washed with ChIP Wash Buffer (50mM Tris-HCl pH 7.5, 250mM LiCl, 10mM EDTA, 1% Triton X-100, 0.5% sodium deoxycholate, protease inhibitors) and with TEN (50mM Tris-HCl pH 8.0, 1mM EDTA, 50mM NaCl). Elution buffer (50mM Tris-HCl pH7.5, 10mM EDTA, 1% SDS) was added and the samples were heated to 65C. Eluates and inputs were digested with 40ug Proteinase K. Both IP and input cross-links were reversed and then samples were phenol:chloroform extracted. DNA was alcohol precipitated and resuspended in TE. ChIP-seq libraries were generated from both IP and Input DNA samples using the NEBNext ChIP-seq Library Prep Master Mix for Illumina with NEBNext Muliplex Oligos for Illumina (NEB). DNA was quantified with both a Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) and a KAPA Library Quantification Kit (KAPA Biosystems). Libraries were size selected using Agencourt AMPure XP resin (Beckman Coulter) and sizes confirmed on a 2100 Bioanalyzer (Agilent).

Sequencing Platform

instrument_model
Illumina HiSeq 2500

mm10

Number of total reads
32544672
Reads aligned (%)
90.6
Duplicates removed (%)
14.8
Number of peaks
10666 (qval < 1E-05)

mm9

Number of total reads
32544672
Reads aligned (%)
90.2
Duplicates removed (%)
16.1
Number of peaks
10713 (qval < 1E-05)

Base call quality data from DBCLS SRA