3xFLAG-Avi tag insertion at Gli3 initiation codon in V6.5
cell type
ES-derived neural progenitors (dorsal)
chip antibody
anti-FLAG M2 clone
chip antibody vendor
Sigma
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.