GSM1821966: Input for Nkx6.1 and Olig2 chip rep2; 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 neural cells
NA
NA
Attributes by original data submitter
Sample
source_name
Neural progenitor cells
strain background
Mixture of C57BL6/J, 129 Sv and Swiss Webster
cell line
Rosa26-Gli1FLAG (YFP3.1 derivative)
cell type
ES-derived neural progenitors (ventral)
chip antibody
none
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Cells were dissociated in trypsin then crosslinked in 1% formaldehyde at room-temperature for 30 minutes. Cells were lysed in lysis buffer (140mM NaCl, 0.5% NP40, 0.25% Triton-X 100, 10% glycerol, 1mM EDTA, 50mM HEPES-KOH, pH7.5) or (1% SDS, 10mM EDTA, 50mM Tris-HCl, pH 8.1) at 4˚C with agitation for 10 minutes then washed with Buffer 2 (200mM NaCl, 1mM EDTA, 0.5mM EGTA, 10mM Tris-Cl pH8.0) for 10 minutes at room-temperature. Chromatin was fragmented by sonication (Branson digital sonifier with microtip) in Buffer 3 (1mM EDTA, 0.5mM EGTA, 10mM Tris-Cl pH8.0). Lysate was cleared by centrifugation and supplemented with NaCl, NaDeoxycholate, and TritonX 100 to the final concentrations of 100mM, 0.1%, and 1%, respectively. Magnetic beads (Dynal/Invitrogen, sheep anti-mouse IgG, anti-rabbit IgG, and Protein G for mouse, rabbit, and goat antibodies, respectively) were blocked with a blocking buffer (5mg/ml BSA in PBS) then incubated with an antibody in the blocking buffer overnight. Unbound antibody was washed away by extensive wash with the blocking buffer. Beads/antibody complex was incubated with the lysate overnight at 4°C. Beads were washed 5 times with RIPA buffer (1% NP40, 0.7% NaDeoxycholate, 0.5M LiCl, 1mM EDTA, 50mM HEPES-KOH, pH7.5). DNA was eluted with elution buffer (1% SDS, 1mM EDTA, 50mM Tris-Cl, pH8.0) at 65˚C for 15 min, and the eluate was incubated further overnight at 65˚C to reverse crosslinking. DNA was purified by phenol/chloroform extraction and ethanol precipitation or by PCR purification kit (Qiagen). All buffers except RIPA buffer was supplemented with protease inhibitor cocktail mix (Roche). Sequencing library was constructed using ChIP-Seq DNA Sample Prep Kit (Illumina IP-102-1001) according to the manufacturer's instruction. Briefly, DNA was blunted and phosphorylated with T4 and Klenow DNA polymerases and T4 PNK followed by adapter ligation with T4 DNA ligase. A range of 275-700bp was selected by gel purification and amplified with PCR primers 1.1 and 2.1 for 18 cycles (Sample 1-8). Sequencing library was constructed using NEBNext Ultra DNA Library Prep Kit for Illumina (New England Biolabs, E7370) according to the manufacturer's instruction with 15 cycles of PCR amplification (Sample 9-13).