GSM5693592: CTL_Sperm_CTCF_Rep2_R1; Mus musculus; ChIP-Seq
Cells were crosslinked with 1% formaldehyde in 1xPBS for 10 min at RT, and the reaction was quenched with 125 mM glycine for 10 min at RT. After washing with PBS, cells were lysed with 5 mM PIPES, 85 mM KCl, 0.5% NP40 and 1x protease inhibitors (P8107S, NEB) on ice for 15 min. After centrifugation, cells were resuspended in RIPA buffer (1x PBS; 1% NP40; 0.5% sodium deoxycholate; 0.1% SDS; 1x protease inhibitors) and incubated on ice for 20 min. The purified chromatin was sonicated to 300-500 bp using a Diagenode Bioruptor. After 25 cycles (30 seconds on and 60 seconds off), the supernatant containing sheared chromatin was collected. Immunoprecipitation was performed overnight at 4°C with antibodies. Libraries for Illumina sequencing were constructed using the following standard protocol. Fragment ends were repaired using the NEBNext End Repair Module and adenosine was added at the 3' ends using Klenow fragment (3' to 5' exo minus, New England Biolabs). Precipitated DNA was incubated with adaptors at room temperature for 1 hr with T4 DNA ligase (New England Biolabs) and amplified with Illumina primers. Two independent biological replicates per sample were then sequenced using paired-end 50 bp on Illumina Hiseq2500 v4 or NovaSeq 6000 instruments.