title: GSM2259908: ES_Serum_Input_ChIPseq_library; Mus musculus; ChIP-Seq
library_construction_protocol: Approximately 20 million cells were fixed in serum-free culture media supplemented with formaldehyde added to a final concentration of 1%. After 10 minute incubation at room temperature, fixation was terminated by adding 2.5M glycine stock to a final concentration of 125 mM glycine. Cross-linking termination was carried out for 5 minutes at room temperature followed by 15 minutes at 4°C. Cells were harvested with silicone scraper and pelleted, washed once with PBS, snap-frozen and stored at -80°C until processing.   Cell pellets were thawed for 10 min on ice before use. Nuclei were isolated by resuspending each pellet in 1 mL Cell Lysis Buffer (10 mM Tris pH 8.0, 10 mM NaCl, 0.2% NP-40/Igepal, Protease Inhibitor, PMSF), incubating on ice for 10 min, and spinning to pellet. Nuclei were resuspended in 500 uL Nuclear Lysis Buffer (50 mM Tris pH 8.0, 10 mM EDTA, 1% SDS, Protease Inhibitor, PMSF) and incubated on ice for 20 min. After bringing the samples up to volume by the addition of 300 uL IP Dilution Buffer (20 mM Tris pH 8.0, 2 mM EDTA, 150 mM NaCl, 1% Triston X-100, 0.01% SDS, Protease Inhibitor, PMSF), samples were sonicated for 45 minutes using an Epishear sonicator set at 100% amplitude, with cycles of 30 seconds on and 30 seconds off. The resulting sheared chromatin was spun down, and the supernatant was transferred to a preclearing solution of 3.7 mL IP Dilution Buffer, 0.5 mL Nuclear Lysis Buffer, 175 uL of Agarose Protein A/G beads, and 50 ug Rabbit IgG, and rotated at 4°C. 35 uL Protein A/G agarose beads were pre-bound with 10 uL anti-CTCF antibody (Millipore #07-729) and incubated for 2 hours during the pre-clear stage. After a two hour pre-clear incubation, the beads were pelleted, and 4.5 mL supernatant was removed. 200 uL was reserved for input control, while the remaining supernatant was transferred to agarose beads pre-bound with antibody and rotated overnight at 4°C. Bound bead complexes were washed once with 1 mL IP Wash Buffer 1 (20 mM Tris pH 8.0, 2 mM EDTA, 50 mM NaCl, 1% Triton X-100, 0.1% SDS), twice with 1 mL High-Salt Buffer (20 mM Tris pH 8.0, 2 mM EDTA, 500 mM NaCl, 1% Triton X-100, 0.01% SDS), once with IP Wash Buffer 2 (10 mM Tris pH 8.0, 1 mM EDTA, 0.25 M LiCl, 1% NP-40/Igepal, 1% Na-deoxycholate), and finally once with 1x TE. Complexes were eluted by twice resuspending bound beads in 110 uL Elution Buffer (100 mM NaHCO3, 1% SDS), pelleting the beads after each elution and transferring 100 uL supernatant to a new tube. Finally, 12 uL of 5M NaCl and 20 ug RNase A were added to both 200 uL IP and input samples and incubated at 65 degrees for 1 hour, followed by the addition of 60 ug of Proteinase K and overnight incubation at 65 degrees. DNA was isolated via phenol-chloroform extraction and ethanol precipitation, and concentration was quantified using Qubit fluorometer. Libraries were prepared for sequencing using the NEBNext Ultra Library Prep Kit (NEB #E7370) and following the manufacturer’s protocol for ChIP-seq library preparation. No size selection step was performed following adapter ligation. The libraries were amplified over 18 PCR cycles using NEBNext Multiplex Oligos for Illumina (NEB #E7335). The final ChIP libraries were eluted in 30 uL 0.1x TE from the Agencourt AMPure XP beads, at which point we confirmed the library contained DNA fragments ranging from 250 to 1200 bp, including the adapters, by running a High-Sensitivity DNA assay on an Agilent Bioanalyzer. The concentration of these libraries was assayed via the KAPA Illumina Library Quantification Kit (#KK4835), diluted to equivalent concentrations and pooled, and finally sequenced with 75-cyles per paired-end on the Illumina NextSeq500.
attributes.tag: GEO Accession
attributes.value: GSM2259908