title: GSM9429372: ChIP-seq, CTCFL antibody, humanized mouse, MEFs, biological replicate 2; Mus musculus; ChIP-Seq
library_construction_protocol: Mice were euthanized via carbon dioxide asphyxiation followed by cervical dislocation, in accordance with institutional animal care guidelines. Tissues were immediately harvested and placed in ice-cold phosphate-buffered saline (PBS). For tissue dissociation, samples were transferred to a sterile dish containing PBS (250 µL per 50 mg of tissue) and finely minced. The minced tissue was then transferred to a 50 mL Falcon tube and further homogenized by repeated passage through an 18-gauge needle (10 times) followed by a 21-gauge needle (20 times). Large debris and clumps were removed using a 70 µm cell strainer. Cells were pelleted by centrifugation and resuspended in 2 mL Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS). To crosslink chromatin (from tissues and MEFs), formaldehyde was added to the cell suspension to a final concentration of 1%, and samples were incubated on a rocker at room temperature for 10 minutes. The reaction was quenched by the addition of glycine (final concentration 125 mM) and incubated for an additional 10 minutes. Cells were washed twice with ice-cold PBS and pelleted by centrifugation. Cell pellets were stored at -80°C until further use. For chromatin immunoprecipitation (ChIP), cells were thawed on ice and resuspended in ChIP lysis buffer (150 mM NaCl, 1% Triton X-100, 0.1% SDS, 20 mM Tris–HCl pH 8.0, 2 mM EDTA). Chromatin was sheared to a fragment size of 200–600 bp using sonication. Immunoprecipitation was performed using 5 µg of the appropriate antibody. Immunoprecipitated DNA was purified using the ChIP DNA Clean & Concentrator Kit (Zymo Research, catalog #D5205). ChIP-seq libraries were prepared using the NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs, catalog #E7645S), following the manufacturer's protocol. Libraries were amplified using 10 cycles of PCR and size-selected to 250–450 bp using AMPure XP beads (Beckman Coulter, catalog #A63881). Library quality was assessed using the Agilent High Sensitivity DNA Kit (Agilent Technologies, catalog #5067-4626) on an Agilent 2100 Bioanalyzer.