GSE121926
H3K27ac ChIP-Seq and mRNA-Seq analysis of five human B-lymphoblastoid cell lines (LCLs) prepared from the same healthy male individual (CEPH/UTAH).
Purpose: We examined the extent and nature of non-genetic variability at the gene regulatory activity and gene expression levels in isogenic LCLs, highlighting features of the LCL model that might aid the rational design of genetic association studies. Brief method: H3K27ac ChIP-Seq: Crosslinked chromatin corresponding to 5 million cells was immunoprecipitated with 2.5 ug anti-histone H3 (acetyl K27) antibody (Abcam, cat. ab4729) or isotype control antibody (Santa Cruz Biotechnology, cat. sc-2027 X). in biological duplicates. Library preparation was performed by the Genomic Medicine and Bioinformatics Core Facility at the University of Debrecen, Debrecen, Hungary from 10 ng of ChIP material based on the “TruSeq ChIP Sample Preparation Guide 15023092 B” with minor modifications. Cluster generation, sequencing (50-bp, single-end) and demultiplexing (bcl2fastq Conversion Software) were performed either at the Genomic Medicine and Bioinformatic Core Facility at the University of Debrecen or at the EMBL Genomics Core Facility, Heidelberg, Germany. BWA 0.7.10 was used to align ChIP-Seq reads to the hg19 (GRCh37) genomic build. HOMER 4.9.1. was used to predict genomic regions with H3K27ac enrichment. We used the R package DiffBind (Bioconductor) to define a consensus set of predicted gene regulatory elements, calculate RPKM values, cluster the samples and create a correlation matrix. RNA-Seq: Total RNA was extracted from two million cells with TRIzolate reagent in biological duplicates. Sequencing libraries were prepared following Illumina's TruSeq RNA Sample Preparation v2 Guide with poly(A) selection using 1 µg total RNA as the starting material. Indexed libraries were pooled and subjected to single-end sequencing on a NextSeq 500 sequencer (Illumina, San Diego, CA, USA) with 50-bp read length. Library preparation, cluster generation, sequencing and base calling were performed at the Genomic Medicine and Bioinformatic Core Facility at the University of Debrecen, Hungary. Demultiplexing was performed using the bcl2fastq Conversion Software (Illumina). RNA-Seq reads were aligned to the hg19 (GRCh37) genomic build using TopHat v2.0.7 (--max-multihits option set to 1). Transcript abundances were calculated and batch effect was accounted for using edgeR and UCSC gene annotation track (hg19, downloaded from Illumina's iGenomes database in 07/17/2015), and are expressed as RPKM values. Genes with CPM values (read counts per million mapped reads) below 5 across all samples were considered unexpressed and were discarded. Results: We found extensive gene regulatory region activity differences between the five isogenic LCLs affecting mostly typical enhancers. clustered enhancers (super-enhancers). H3K27ac variability is coupled with a modest gene expression variability. The set of variable genes was enriched for genes involved in lymphocyte-specific signaling pathways and drug response. Principal investigator: Balint L. Balint, lbalint@med.unideb.hu Overall design: H3K27ac ChIP-Seq and mRNA profiles of five LCLs in biological duplicates.