GSM6081036: RNA_Clone2.12_4; Homo sapiens; RNA-Seq
We performed anti-TFAP2A, anti-MITF, anti-H3K27Ac, anti-H3K4Me3 and anti-H3K27Me3 Cleavage Under Targets and Release Using Nuclease (CUT&RUN) sequencing and ATAC-Sequening in wild-type and TFAP2A; TFAP2C double mutant SK-MEL-28 cell lines essentially as described (Skene and Henikoff 2017). CUT&RUN libraries were prepared using the KAPA Hyper Prep Kit (Roche). Quality control post-library amplification was conducted using the 2100 Bioanalyzer for fragment analysis. Libraries were pooled to equimolar concentrations and sequenced with paired-end 150 bp reads on an Illumina NovaSeq 6000 instrument. ATAC-seq was performed according to (Buenrostro et al., 2015; Liu et al., 2020) with minor alterations. Briefly, 70,000 TFAP2-KO cells (clone 2.12 and clone 4.3, four replicates each) and WT cells (four replicates) were lysed in ice-cold lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% NP-40: Sigma). Transposition was performed directly on nuclei using 25 µl tagmentation reaction mix (Tagment DNA Buffer #15027866, Tagment DNA Enzyme #15027865 from Illumina Tagment DNA kit #20034210). Tagged DNA was subjected to PCR amplification and library indexing, using the NEBNext High-Fidelity 2x PCR Master Mix (New England Biolabs #M0451S) with Nextera DNA CD Indexes (Illumina #20015882), according to the following program: 72 ̊C for 5 minutes; 98 ̊C for 30 seconds; 12 cycles of 98 ̊C for 10 seconds, 63 ̊C for 30 seconds, and 72 ̊C for 1 minute. The PCR product was purified with 1.8 times the volume of Ampure XP beads (Beckman Coulter #A63881). Library quality was assessed using a BioAnalyzer 2100 High Sensitivity DNA Chip (Agilent Technologies). All DNA libraries that exhibited a nucleosome pattern were pooled and processed for 150bp paired-end sequencing. RNA-seq was performed on four replicate RNA-seq experiments were performed on TFAP2-KO cells (clone 2.12 and clone 4.3) and WT cells. Total RNA was extracted by direct cell lysis using the RNeasy Plus Mini Kit with QiaShredder (Qiagen #47134). RNA samples with an RNA integrity number (RIN) above nine were used for library generation and 150 bp paired-end sequencing on the Illumina HiSeq2500 platform (Novogene, Sacramento, CA). FASTQ sequence files were processed using FastQC (Babraham Bioinformatics) for quality control, and reads were trimmed using Trim Galore Version 0.6.3 (Developed by Felix Krueger at the Babraham Institute) and subsequently aligned to human genome assembly hg19 (GRCh37) using STAR. The output of the --quantMode GeneCounts function of STAR was used for the calculation of differential transcript expression using DESeq2 [1].The rlog function was used to generate log2-transformed normalized counts. Adjusted p-value < 0.05 was used as the threshold for statistically significant differences.