GSM7881745: D4_TKO_CTCF_CUTnRUN_rep1; Mus musculus; OTHER
We performed CUT&RUN according to the original protocol (Skene and Henikoff 2017) with the following modifications: 500,000 cells were used for each sample, and the primary antibody was incubated overnight at 4ºC on a rotator. After incubation with pAG-MNase and performing the MNAse reaction, the samples were placed on a magnetic rack and the supernatant containing the DNA samples was recovered. Following addition of 0.1% SDS and 0.17mg/ml Proteinase K, samples were incubated at 50°C for 1h. Purified DNA was obtained by phenol/chloroform extraction and precipitated with 100% Ethanol by centrifugation. The DNA pellet was washed in 80% ethanol, spun down and air-dried before being resuspended in 25µl of 1mM Tris-HCl pH8.0. We used a primary CTCF antibody (Cell D31H2) or IgG control (SigmaAldrich I5006) and no secondary antibody was used for these experiments. The pAG-MNase plasmid was obtained from Addgene (#123461), and the protein was purified by the Curiecoretech Recombinant Protein Platform. Sequencing library preparation was made using the NEBNext® UltraTM II DNA Library Prep Kit for Illumina (NEB) following the procedure described in “Library Prep for CUT&RUN with NEBNext® UltraTM II DNA Library Prep Kit for Illumina® (E7645) V.1” available in protocols.io. Quality control for the finalized libraries was performed using a TapeStation 420 system (Agilent). Libraries were sequenced by Novogene Co on a NovaSeq using paired-end 150 base pair parameters, requesting 4 gigabytes of data per sample, or approximately 13 million reads.