GSM2065701: input-of-ChIP-in-Sall4KO+WT; Mus musculus; ChIP-Seq
Cells for ChIP assays were crosslinked in 1% formaldehyde for 8-10 min at room temperature. The reaction was terminated by adding 2 M glycine to a final concentration of 125 mM. After being washed with PBS, the cells were resuspended in lysis buffer 1 (50 mM HEPES, pH 7.9, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% IGEPAL CA-630 and 0.25% Triton X-100) and incubated for 10 min on ice. After centrifugation, the cells were resuspended in lysis buffer 2 (10 mM Tris-HCl, pH 8.0, 200 mM NaCl, 1 mM EDTA and 0.5 mM EGTA) and incubated for 10 min at room temperature. The chromatin fraction was resuspended in lysis buffer 3 (10 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% sodium deoxycholate and 0.5% sodium N-lauroyl sarcosine) and sonicated into 200-400 base-pair (bp) fragments (Bioruptor, Diagenode). Chromatin fragments were immunoprecipitated overnight with antibodies against SALL1, SALL4A and TET1. The protein-antibody complexes were incubated with pre-washed Dynabeads Protein A or G (Life Technologies) for 1 hr. The beads were sequentially washed with the following buffers: once with FA-lysis buffer (150 mM NaCl, 1% Triton X-100, 50 mM HEPES, pH 7.9, 2 mM EDTA and 0.5% sodium deoxycholate), twice with FA high salt buffer (500 mM NaCl, 1% Triton X-100, 50 mM HEPES, pH 7.9, 2 mM EDTA and 0.5% sodium deoxycholate), once with LiCl buffer (250 mM LiCl, 0.5% IGEPAL CA-630, 0.5% sodium deoxycholate, 1 mM EDTA and 10 mM Tris-HCl, pH 8.0) and twice with TE buffer (10 mM Tris-HCl, pH 8.0 and 1 mM EDTA). Protein complexes were eluted by incubation with 250 μl of freshly prepared elution buffer (1% SDS and 100 mM sodium bicarbonate) for 15 min at room temperature. The elution step was repeated once, and the eluted fractions were combined.  For ChIP of bio-SALL4A, chromatin fragments were incubated with Dynabeads M-280 Streptavidin (Life Technologies) equilibrated with PBS + 1% BSA. Triton X-100 was added to a final concentration of 1%. Protein-bound beads were washed sequentially with following buffers: 2% SDS twice, FA high salt buffer twice, LiCl buffer (250 mM LiCl, 1% IGEPAL CA-630, 0.5% sodium deoxycholate, 1 mM EDTA and 10 mM Tris-HCl, pH 8.0) once and TE buffer twice. Protein complexes were eluted by incubating the beads in 300 μl of elution buffer at 65℃ with agitation at 800 rpm overnight.  After crosslinking reversal and RNase A and proteinase K treatment, DNA was extracted with phenol-chloroform and precipitated with ethanol.  For DNA immunoprecipitation assays, genomic DNA was isolated by conventional phenol extraction and proteinase K treatment. The purified genomic DNA was sonicated into 200-400 bp fragments. Adapter-ligated DNA libraries were constructed. DNA fragments were denatured in 0.4 M NaOH by incubation at 95℃ for 10 min and then placed on ice immediately and neutralised by adding an equal volume of 2 M ammonium acetate. Samples (3 μg) of the adapter-ligated DNA libraries were immunoprecipitated with 5 μg of mC or hmC antibodies, and 5 μg of DNA with 0.2 μl of caC anti-serum.  For mRNA deep sequencing analysis, total RNA was isolated by TRIzol Reagent (Life Technologies), and mRNA with a poly (A) tail was purified and subjected to sequencing library construction. The ChIP-seq libraries were constructed using NEBNext DNA Sample Prep Master Mix (NEB).DNA libraries were subjected to size-selection using AMPure XP beads (Beckman Coulter) and PCR amplification (18-20 cycles for ChIP-seq DNA libraries and 12-16 cycles for DIP-seq libraries).
GEO Accession
GSM2065701