RNA sequencing of EHMT1 haploinsufficient hiPSC-derived astrocytes

Vermeulen-Kalk, K.
Wang, S.
Kummeling, J.
Mossink, B.
Frazier, Z.
Rozumny, B.
O’Donnell-Luria, A.
Hanson, E.
Frega, M.
Linda, K.
Negwer, M.
Lendemeijer, B.
Oudakker, A.
Pop-purceleanu, M.
Janzing, J.
van Dongen, L.
de Vrij, F.
Kushner, S.
van der Werf, I.
Schoenmaker, C.
Oomens, W.
Srivastava, S.
Egger, J.
Wijnant, K.
Puvogel , S.
van Bokhoven, H.
Schubert, D.
Nadif Kasri, N.

Kleefstra syndrome (KLEFS1) results from EHMT1 haploinsufficiency and is characterized by variable neurodevelopmental delays and psychopathology. To investigate the downstream effects of EHMT1 haploinsufficiency in human astrocytes, and thereby their potential involvement in KLEFS1 pathobiology, we performed transcriptomic profiling of EHMT1⁺/⁻ human induced pluripotent stem cell (hiPSC)-derived astrocytes. This repository contains RNA sequencing (RNA-seq) data, including both raw FASTQ files and processed gene-level count tables, generated from wild-type (WT; control) and CRISPR/Cas9-edited EHMT1⁺/⁻ hiPSC-derived astrocytes. Each condition includes three technical replicates. RNA isolation and quality control: RNA was extracted from hiPSC-derived astrocyte cultures at four weeks of differentiation in astrocyte medium using the NucleoSpin RNA Isolation Kit (Macherey-Nagel, 740955.250). RNA yield was quantified using a NanoDrop spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). All samples had an RNA Integrity Number (RIN) > 9. Library preparation and sequencing: Strand-specific cDNA libraries were prepared with the NEBNext Ultra II Directional RNA Library Prep Kit. Strandedness was reversed during insertion of dIDT UMI adapters. Libraries were sequenced on an Illumina NovaSeq 6000 platform (GenomeScan B.V., Leiden), generating approximately 40 million paired-end reads per sample. In the resulting libraries, Read 1 (R1) corresponds to the sense strand, Read 3 (R3) to the antisense strand, and Read 2 (R2) contains the unique molecular identifier (UMI), which was transferred to the RX tag in the BAM files for UMI-aware deduplication. Data processing: Adapter and quality trimming were performed using fastp. Reads were aligned to the GRCh38 reference genome using HISAT2 with the --rna-strandness FR option. Resulting BAM files were sorted and indexed using SAMtools, followed by UMI-based deduplication with UMI-tools. Gene-level quantification was performed with featureCounts (Subread package). Note: Sample WT_1 was sequenced in two separate runs; therefore, two sets of FASTQ files (R1, R2, R3) are provided for this sample. Data files: -R1, R2, and R3 FASTQ files. -all_featurecounts.txt: Gene-level count matrix for all samples. -Metadata.xlsx: Detailed sample metadata, including cell line, organism, cell type, genotype, origin (control or CRISPR), and file identifiers. Metadata formatting adheres to standard public RNA-seq data submission guidelines.