title: Bulk RNA-seq of human skin organoid (SkO), untreated, replicate 2
design: For bulk RNA-sequencing 4 SkOs per condition (untreated, 2 days post infection (dpi), 4dpi, 6dpi, 8dpi) were used. Total RNA was isolated individually from each SkO by adding QIAzollysis reagent (Qiagen) and disrupting the organoid by grinding with a pestle. The lysate was homogenized with a QIAshredder (Qiagen). After one round of chloroform extraction, RNA was cleaned up with the RNeasy MinElute Cleanup Kit (Qiagen). DNA was two times digested with the DNA-free DNA removal kit (Thermo Fisher Scientific). RNA concentration was determined with the Qubit RNA broad range assay (Thermo Fisher Scientific) and equal amounts of RNA from two SkOs of the same condition were pooled resulting in duplicates for each condition for sequencing. RNA integrity was determined via a Bioanalyzer RNA 6000 Pico Assay (Agilent, 5067-1514). RIN values were in the range between 5.5 and 9.2. From each pooled sample 520 ng total RNA was subjected to rRNA depletion (following the Lexogen RiboCop rRNA Depletion Kit for Human/Mouse/Rat V2 protocol, catalog number: 144.24) and further processed via the RNA-Seq V2 Library Prep Kit with UDIs (Lexogen, catalog number: 175.96) in the short insert size variant (RTM) according to manufacturers instructions. Libraries were quality controlled on a Bioanalyzer High Sensitivity DNA Assay (Agilent, 5067-4626) and were sequenced on an Illumina NextSeq 500 instrument with NextSeq 500/550 High Output Kit v2.5 (75 Cycles, product number: 20024906) in a single-read mode (1 x 83 bp). After demultiplexing via bcl2fastq, 20.7  28.6 million (M) read pairs assigned to each sample. All samples passed quality control by fastqc and were subjected to downstream analysis. Processing was done with the nf-core/RNA-seq pipeline (v3.12.0) within the nf-core framework 45. The pipeline was executed with Nextflow v23.04.1 46, utilizing STAR (v2.6.1d)47 for alignment to the GRCh38 human genome and Human herpesvirus 1 genome (X14112.1), as well as Salmon (v1.10.1)48 for transcript-level quantification based on STARs alignment output. To eliminate duplicate reads, the --with_umi option was enabled. Gene-level quantification was derived from transcript-level estimates using the R package tximport (v1.34.0)49, which sums transcript abundances for each gene. The generated raw gene-level counts were normalized and variance-stabilized using the regularized log transformation (rlog) from DESeq2 (v1.34.0)50, addressing the variance-over-mean trend. Principal component analysis (PCA) confirmed high reproducibility across biological replicates. Differential expression analysis was performed using DESeq2, identifying genes as significantly differentially expressed (DEGs) if they exhibited a fold change  2 and an adjusted P-value  0.05. The normalized rlog-transformed counts were then used for clustering and visualization. An overrepresentation analysis of differentially expressed host genes (padj. 0.05 and log2FC 1/-1) was performed with WebGestalt51. GO terms associated with biological processes were selected with an FDR0.05.
identifiers.namespace: SUB15824966
identifiers.id: LIB02