GSM9093384: vKO_DP_Ctcf_rep1; Mus musculus; OTHER
DP thymocytes from WT and Satb1 KO mice were used for CUT&RUN using the benchtop protocol. One million cells were sorted and washed in ice-cold phosphate-buffered saline (PBS). Nuclei were isolated by hypotonic lysis in 1 ml NE1 (20 mM HEPES-KOH pH 7.9; 10 mM KCl; 1 mM MgCl 2 ; 0.1% Triton X-100; 20% Glycerol) for 5 min on ice followed by centrifugation as above. Nuclei were briefly washed in 1.5 ml Buffer 1 (20 mM HEPES pH 7.5; 150 mM NaCl; 2 mM EDTA; 0.5 mM Spermidine; 0.1% BSA) and were incubated with pre-washed Concanavalin A magnetic beads on RT for 5 min. Nuclei-bound beads were washed in 1.5 ml Buffer 2 (20 mM HEPES pH 7.5; 150 mM NaCl; 0.5 mM Spermidine; 0.1% BSA). Nuclei were resuspended in 500 μl Buffer 2 and 10 μl SMC1A antibody (Abcam) or CTCF (Cell Signaling Technologies) was added and incubated at 4°C for 2 hr. Nuclei were washed 3 x in 1 ml Buffer 2 to remove unbound antibody. Nuclei were resuspended in 300 μl Buffer 2 and 5 μl pAG-MNase (EpiCypher) added and incubated at 4°C for 1 hr. Nuclei were washed 3 x in 0.5 ml Buffer 2 to remove unbound pA-MN. Tubes were placed in a metal block in ice-water and quickly mixed with 100 mM CaCl2 to a final concentration of 2 mM. The reaction was quenched by the addition of EDTA and EGTA to a final concentration of 10 mM and 20 mM respectively. Cleaved fragments were liberated into the supernatant by incubating the nuclei at 4°C for 1 h, and nuclei were pelleted by centrifugation as above. DNA fragments were extracted from the supernatant and used for the construction of sequencing libraries. he NEB-Next UltraII library prep kit (NEB) was utilized to prepare a library with an equivalent quantity of DNA (~5 ng) as input. Quantitative PCR (qPCR) analysis was used to estimate the number of cycles required for the amplification of adapter- ligated libraries. Using the HiPrep PCR clean up system (MagBio Genomics, USA), final libraries were purified. Prior to pooling libraries at an equimolar ratio, the library concentration was ascertained using Qubit, and the average fragment size was measured using the dsDNA HS test on the Bioanalyzer 2100 (Agilent). The HiseqX platform was utilized to acquire sequencing reads (150 bp PE).