Lung squamous cell carcinoma from Lkb1fl/fl;Ptenfl/fl (LP) mouse model
tissue
Lung squamous cell carcinoma from Lkb1fl/fl;Ptenfl/fl (LP) mouse model
time
Flash-frozen
chip antibody
None
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
20 million cells per ChIP were cross-linked in 1% formaldehyde for 10 min at room temperature. Crosslinking was stopped with 125 mM glycine and nuclei were extracted. Alternatively, flash-frozen tumors were directly used. Chromatin was sonicated using an Epishear Probe Sonicator (Active Motif) for 4 min at 40% power. SOX2 antibody (R&D Systems Cat#AF2018) was used for IP. Libraries were prepared using a standard Illumina library construction protocol. Briefly, DNA was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (exo minus) and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3' end. After adapter ligation DNA was PCR amplified with Illumina primers for 15 cycles and purified using Ampure XP beads.