GSM1144550: WT MES INP rep1; Mus musculus; ChIP-Seq
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
Input control
Antigen
Input control
Cell type
Cell type Class
Pluripotent stem cell
Cell type
mESC derived mesodermal cells
NA
NA
Attributes by original data submitter
Sample
source_name
ES cell derived day4 EB (embryoid body) Flk1+ mesodermal cells
cell line
WT J1 mESC
cell type
Flk1+ mesodermal cells
treatment
EB formation, enrichment for Flk1+ cells at day 4
chip antibody
N/A (input sample)
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Cells were trypsinized, washed with PBS and crosslinked with 1% formaldehyde in PBS for 10 min at RT. After PBS washing, cells were resuspended in 400 μl of lysis buffer (1% SDS, 20 mM EDTA and 50 mM Tris-HCl (pH 8.0)) containing protease inhibitors (Roche, Indianapolis, IN), incubated for 10 min on ice and sonicated using Misonix cup-horn sonicator to achieve, on average, 200bp fragments for ChIP-seq and 500bp fragments for ChIP-PCR. The lysate was diluted 10 times with ChIP dilution buffer containing 0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA and 16.7 mM Tris-HCl (pH 8.1) and immunoprecipitatied with 4 ug of corresponding antibody overnight at 4 degrees. 20 μl of the lysates were used as input. The complexes were captured using protein A Dynabeads (Invitrogen, Grand Island, NY) and washed twice with the following buffers: low-salt immune complex wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl (pH 8.1); high-salt immune complex wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl (pH 8.1) and 500 mM NaCl); LiCl wash buffer (0.25 M LiCl, 1% NP40, 1% deoxycholate, 1 mM EDTA and 10 mM Tris-HCl (pH 8.1)) and TE (10 mM Tris-HCl and 1 mM EDTA (pH 8.0)). After elution with 50 mM Tris-HCl, pH 8.0, 1 mM EDTA, and 1% SDS, crosslinks were reversed by overnight incubation at 65°C. Samples were then treated with RNase A for 30 min at 37°C and proteinase K for 2 h at 56°C. DNA was subsequently purified using Qiagen MinElute Columns according to manufacturers instructions. DNA concentration was measured using a Qubit (Invitrogen, Grand Island, NY). The library for sequencing was constructed using Ovation Ultralow IL Multiplex System 1-8 according to manufacturer's instructions (Nugen, San Carlos CA). Libraries were sequenced using HIseq-2000 (Illumina, San Diego, CA) to obtain 50 bp long reads.