GSM5419299: Brd4-CUT&Tag in naïve CD8+T cells_rep1; Mus musculus; OTHER
Fifty-thousand isolated CD8+ T cells were used to generate CUT&Tag library as a reference[31]. First, CD8+ T cell was incubated with primary antibody against Brd4 (1:50; Active motif AB_2615059) and then with guinea pig anti-rabbit secondary antibody (1:100; Antibodies Online ABIN101961). This was followed by adding the prepared pA-Tn5 complex. Next, DNA was fragmented by Mg2+ activator and the resulting DNA segments were extracted to amplify with PCR. Finally, PCR products were cleaned up and sequenced. Paired-end 150-bp sequencing was performed on an Illumina HiSeq 2500. Each DNA library for CUT&Tag was sequenced with HiSeq 2500 system (Illumina, USA) under the paired-end 150 bp mode. For data processing, raw sequencing data were trimmed and filtered by using Trim Galore. The paired-end reads with high quality (Q30) were aligned to the mouse reference genome mm10 using the Burrows Wheeler Aligner with default parameters and then sorted using the SAMtools software. The “MarkDuplicates” function embedded in Picard was used to mark and discard PCR duplicates. For comparison between different samples, mapped reads were down-sampled to equalize reads across samples by using Samtools. MACS2 was used to quantify the Brd4 CUT&Tag signal throughout the genome and within gene bodies.