Transcriptome profiling (RNA-seq) of CREBBP+/+ and CREBBP+/- clones of U2932 DLBCL cell line

Purpose: Diffuse large B cell lymphomas (DLBCL) frequently harbor mutations in the histone acetyltransferase CREBBP, however their functional contribution to lymphomagenesis remains largely unknown. This study aims at elucidating and characterizing the molecular pathways affected by mutations in CREBBP. Methods: U2932, a DLBCL cell line that has wild type expression of CREBBP was manipulated by CRISPR-Cas9 strategy to mutate one allele of CREBBP and examine the pathways affected. RNA was isolated using the NucleoSping RNA Kit (Macherey-Nagel) from five wild type (CREBBP+/+) and five heterozygous clones (CREBBP+/-). RNA quality was assessed by Bioanalyzer 2100 followed by library preparation using the TruSeq RNA Sample Prep Kit v4 (Illumina). Sequencing was subsequently performed on the Illumina HiSeq 2500 instrument. RNA-seq reads were quality-checked with fastqc, which computes various quality metrics for the raw reads. RNA-seq reads were mapped to the GRCh38 reference human genome using STAR and reads were counted according to Ensembl gene annotation using the featureCounts function in the Rsubread Bioconductor package. Statistical analysis of differential expression was conducted with the DESeq2 package. Overall design: Trascriptomic profiles of CREBBP+/+ and CREBBP+/- clones were generated by deep sequencing.

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Hashwah H, Schmid CA, Kasser S, Bertram K, Stelling A, Manz MG, Müller A