title: GSM7213858: ESC_CRY1_ChIP; Mus musculus; ChIP-Seq
library_construction_protocol: Cells were cross-linked with 1% Formaldehyde for 10 min at room temperature. Collected cells were resuspended in cell lysis buffer (5 mM HEPES, pH 8.0, 85 mM KCL, 0.5% NP-40, and the same inhibitors as described in the Western blot section) and incubated for 10 min at 4oC. Samples were spun down and the pellets were resuspended in RIPA buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, pH 8.0, 1% Triton X-100, 0.1% SDS, 0.1% sodium deoxycholate, and the same inhibitors for cell lysis buffer) and incubated for 20 min at 4oC.  Nuclear samples were sonicated to fragment DNA to ~0.2 kb using Bioruptor (Diagenode). Sheared DNA was pre-cleared for 2 hr at 4oC with protein G or A sepharose beads (Sigma-Aldrich) and immunoprecipitated with anti-CRY1 antibody (MBL Life science, PM081), overnight at 4oC. Protein A beads were added and rocked for 3hr at 4oC. The beads were washed with low-salt RIPA (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, pH 8.0, 1% Triton X-100, 0.1% SDS, and 0.1% sodium deoxycholate), high-salt RIPA (50 mM Tris-HCl, pH 8.0, 500 mM NaCl, 1 mM EDTA, pH 8.0, 1% Triton X-100, 0.1% SDS, and 0.1% sodium deoxycholate), LiCl wash (10 mM Tris-HCl, pH 8.0, 250 mM LiCl, 1 mM EDTA, pH 8.0, 0.5% NP-40, and 0.1% sodium deoxycholate), and TE buffer (10 mM Tris-HCl, pH 8.0 and 1 mM EDTA, pH 8.0). Samples were reverse-crosslinked in elution buffer (10 mM Tris-HCl, pH 8.0, 300 mM NaCl, 5 mM EDTA, pH 8.0, and 0.5% SDS) overnight at 65oC, followed by the treatment with RNase for 30 min at 37oC, and proteinase K for 1 hr at 55oC. DNA was purified by phenol/chloroform and eluted in water. Anti-guinea pig IgG (MBL Life science, PM067) antibody was used as a negative control. Library construction was performed using the Perkin Elmer NEXTFLEX Rapid DNA-seq v2.0 and the Perkin Elmer NEXTFLEX Unique Dual Index barcodes. The DNA quantity was measured by Qubit DNA High Sensitivity and the fragment size was measured by Agilent Bioanalyzer 2100 High Sensitivity DNA chip assay. The input amount of DNA was 2ng. The ends of the DNA were repaired and adenylated. The reaction mixture was then cleaned up using AMPure XP magnetic beads and Illumina-compatible barcoded adapters were ligated onto the blunt-end/adenylated product. The adaptor-ligated product was cleaned using AMPure XP beads and then amplified for adaptor-ligated products using 11 cycles of PCR. The resulting library was cleaned with AMPure XP beads and quantified by qPCR with the Roche Kapa Universal/Illumina Library Quantification kit. The library size was determined by analysis using the Bioanalyzer 2100 DNA High Sensitivity Chip.