RNA concentration was estimated using the Agilent High Sensitivity RNA Screen Tape System, and libraries were prepared using the SMART-Seq® v4 Ultra® Low Input Kit (Takara Bio) with 5 ng total RNA input per sample, as per manufacturer recommendations. cDNA libraries were subject to high-throughput sequencing (Illumina NovaSeq) and ~50 million paired-end reads were generated per sample. Reads were checked for quality (FastQC v0.11.5) and processed using the Digital Expression Explorer 2 (DEE2) workflow55 . Adapter trimming was performed with Skewer (v0.2.2)56 (link). Further quality control was performed with Minion, part of the Kraken package57 (link). Filtered reads were mapped to the mouse reference genome GRCm38 using STAR aligner58 (link) and gene-wise expression counts were generated using the “-quantMode GeneCounts” parameter. The R package edgeR was used to calculate FPKM59 (link).
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