DNA/RNA was extracted using the AllPrep DNA/RNA FFPE Kit (Cat# 80234, Qiagen) following the manufacturer’s instructions. Sample preparation was performed using the NEB NEBnext Ultra Low Input Total RNA with rRNA Depletion kit. The average RNA extracted from SRC and NEP sections was 152.77ng (90.0-318.0) and 188.57ng (90.0-390.0), respectively. cDNA libraries were sequenced on the NovaSeq 6000 platform using paired-end sequencing with 100 bp read lengths. Reads were trimmed to remove sequencing adapters using Cutadapt [12 ] (v1.18), and aligned to the human reference genome hg38 using STAR [13 (link)] (v 2.7.0f) in two-pass mode. Library preparation and sequencing generated between 165 and 255 million reads per sample. More than 91% of the bases were above a quality score of Q30.
RNA expression levels were quantified using RSEM [14 (link)] (v.1.3.1) with GENCODE annotation (v.30) [15 (link)]. Genes with fewer than 10 counts were removed prior to further analysis. Normalization and differential expression analysis was performed using DESeq2 [16 (link)] (v1.32.0) in R (v4.2.2).
RNA expression levels were quantified using RSEM [14 (link)] (v.1.3.1) with GENCODE annotation (v.30) [15 (link)]. Genes with fewer than 10 counts were removed prior to further analysis. Normalization and differential expression analysis was performed using DESeq2 [16 (link)] (v1.32.0) in R (v4.2.2).