Novaseq

CUT&Tag assay was performed as previously described (61 (link)). Briefly, 100,000 WT and FBXO42 KO cells were collected and lysed according to the manufacturer’s guidance (catalog no. 12597, YEASEN). Cell lysates were incubated at room temperature with concanavalin A–coated magnetic beads for 1 hour and then with the primary antibody against RBPJ (1:50; ab25949, Abcam) for 2 hours, with secondary antibodies for 1 hour, and with pA/G-Tn5 adapter complex for 1 hour. The tagmentation takes 1 hour, and DNAs were extracted using a DNA purification kit. Libraries were prepared using the Hieff NGS Tagment Index Kit for Illumina (96 index) (catalog no. 12610, YEASEN) and pooled together for paired-end 150–base pair (bp) sequencing on a NovaSeq (Novogene). Raw fastq files were trimmed using Trim Galore and aligned to the human genome (hg38) using Bowtie2. Reads were sorted and converted to BAM format, and data track visualization occurred using Integrative Genomics Viewer (IGV). Final data analysis and visualization were performed using in-house R scripts.

NovaSeq (30 times genome coverage) was performed by Novogene (Beijing, China). Illumina paired-end reads were trimmed with Trimmomatic v.0.36 (38 (link)), and read quality was assessed with FastQC v.0.11.8. The plasmid sequence was then appended to the human genome sequence downloaded from GENCODE (GRCh38/hg38) (39 (link)), and reads were aligned with the modified human genome sequence with BWA 0.7.17 (40 (link)). Quality control on the raw alignment data was performed by filtering unqualified reads, including unmapped reads and multi-mapped reads, with SAMtools v.1.7.2 (41 (link)). PCR-duplicated reads were identified and removed with Picard v.2.16.0 (42 (link)). Paired-end reads with one read located on the plasmid and the mate read located on human chromosomes were retrieved from the alignment results, and Integrative Genomics Viewer (IGV) v.2.3.34 (43 (link)) was used to evaluate the mapping quality of these soft-clipped reads. To determine the direction of insertion, forward and reverse insertion reference sequences were constructed in chr7:5527490–5527491 for the alignment of paired-end reads.