RNA-seq, ChIP-seq and single cell RNA-seq of human skin Langerhans cells

Langerhans cells (LCs) in the epidermis promote immune homeostasis, efficiently activating tolerogenic and immunogenic T cell responses. To understand genomic programming in human Langerhans cells we performed whole transcriptome (bulk RNA-seq and single cell RNA-seq) profiling and analysis of H3K4Me3 and H3K27Ac histone modifications across LC genome in primary human cells from 6 independent donors. Primary LCs were either unstimulated and stimulated with TNF-alpha. Additionally we performed a CRISPR editing experiment for IRF4 Primary human skin Langerhans cells from independent 3 donors were isolated by migration from epidermal sheets, and either immediately processed for sequencing or exposed to TNF-alpha signalling (2h and 24h). Matched samples from donors LC1 to LC3 were used for time-course stimulation bulk RNA-sequencing, H3K4Me3 and H3K27Ac ChIP sequencing. Additionally 150 unstimulated cells from donor 4 and 1000 unstimulated cells from donor 5 were subjected to single cell RNA-sequencing following encapsulation with Drop-seq. For IRF4 editing experiment primary human migrated LCs were subjected to nucleofaction with purified S. pyogenes Cas9 (spCas9) complexed with a modified single guide RNAs (sgRNAs) targeting IRF4. scRNA-sequencing using DropSeq encapsulation was carried out on 1000 control and 1000 edited primary LCs cells from donor 6 at 48h time point.