The long non-coding RNA ELDR suppresses tumorigenicity of AML cell lines with MLL rearrangements by interfering with DNA replication and chromatin accessibility [ATAC-Seq]
ATAC-seq experiments were performed using the C1 and C3 stable ELDR overexpressing THP1 cell lines, an empty vector control cell line (EV) and non-transfected THP-1 cell (NT). The ATAC-seq experiment was done with two replicas for the two ELDR lines C1 and C3, EV- and NT control each. Overall design: ATAC-seq was carried out with 50,000 cells washed once with 50µl of cold 1X PBS buffer. Spin down at 500g for 5 min, 4 °C. Gently pipetted to resuspend the cell pellet in 50µl of cold lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% IGEPAL CA-630). Cells were spun down immediately at 500g for 10 min, 4°C and lysed with non-ionic detergent to enrich nuclei. Pellets of 50,000 nuclei were tagmented for 30 minutes at 37°C (TDE1 Transposase, Illumina) and tagmented DNA was purified on column DNA clean-5 (Zymo Research). Illumina UDP Indexes were integrated by PCR-enrichment (11 cycles) and final libraries were purified and size selected (L=1.1; R=0.6) with ampure beads (KAPA, Roche Diagnotics). Library size distribution was assessed on a 2100 bioanalyzer (Agilent Technologies) and libraries were quantified by qPCR. Equimolar libraries were sequenced in paired end reads (PE100), on a Novaseq 6000 system (Illumina), with a S4 flow cell and a coverage of 70M fragments per library. The quality of the raw reads was assessed with FASTQC v0.11.8. After examining the quality of the raw reads, trimming was performed with TRIMMOMATIC v0.36.. The reads were aligned to the GRCh38 reference genome (release 102) with BOWTIE2 v2.2.6 with mean of 77 % of reads uniquely mapped. Alignments were post-processed to remove PCR duplicates (Picard tool v2.4.1) and reads mapping to mitochondrial DNA (samtools v1.8). In order to represent the real Tn5 transposase binding sites of 9bp, the coordinates of the reads were shifted by +4bp for the plus strand and by -5bp for the minus strand, using Deeptools v3.0.1. The former was also used to remove ENCODE's blacklisted regions (signal artefact regions) and convert Bam files to BEDPE format. MACS2 was used to identify significant peaks. Diffbind v2.10.0 R package was used to generate the count matrix of Tn5 insertion site numbers for each consensus peak (peaks that were present in all samples for each condition). Differentially accessibility regions (DARs) were identified between conditions within each cell type and controlling for the effect of gender, using DESeq2 v1.30.0 R package. DARs were annotated with their closest feature and different transcription binding motifs identified with HOMER v4.8.0. A non biaised motif search was also performed with HOMER, in order to identify all known motifs and de novo motifs in these regions.