GSM7793707: WT_hom_testis_input.ChIPseq_rep1; Mus musculus; ChIP-Seq
50-100mg of lightly chopped testits tissue was fixed with formaldehyde before quenching with glycine and then needle homogenized. The tissue slurry was pelleted and then lysed with 100ul of Lysis Buffer per 10mg of tissue. Using the Bioruptor Pico sonicator (Diagenode), we sheared lysates and spun the sonicated lysates at max speed for 10min at 4°C to remove cell debris and then rotated them for 2hr at 4°C with 20ul ChIP-grade Protein A/G Magnetic Beads (Pierce) to preclear the lysate. From each chromatin preparation, a 1% volume aliquot was set aside as chromatin input. Pull downs were done with 10ul CTCF (3418s, Cell Signaling Technologies) with overnight rotation at 4°C. Next, we added 20ul Pierce ChIP-grade Protein A/G Magnetic Beads (Pierce) and let samples rotate for 2hrs at 4°C. We then washed the beads consecutively with TBST buffer, Lysis buffer and 1xTE pH=8. Following washes, we eluted chromatin and incubated the ChIP and input samples overnight at 65°C in presence of NaCl to reverse crosslinks and then digested them consecutively with 8ug RNAse A and 80ug Proteinase K. Lastly, we purified the samples using traditional phenol:chloroform extraction and ethanol precipitation. ChIP-seq libraries were contructed using the NEBNext UltraII kit according to manufactor's instructions.