ABSTRACT: Purpose: To identify how follistatin (FST) and LIN28B re-activation augments the regenerative competence of cochlear supporting cells (SCs), we used bulk-RNA sequencing to analyze the transcriptome of SC-containing control, FST, LIN28B and FST+LIN28B overexpressing organoids after 7 days of culture. The 7 day time point was chosen as it correlates with the peak of SC proliferation and presumed SC reprogramming. Methods: Samples were processed using Illumina's TruSeq stranded Total RNA kit, using the UDI indexes. The samples were sequenced on the NovaSeq 6000, paired end, 2x50 base pair reads.We used kallisto (v0.46.1) to pseudo-align reads to the reference mouse transcriptome Ensembl Mus musculus v96 and to quantify transcript abundance. The companion tool sleuth was used to determine differentially expressed genes (DEGs) comparing control to LIN28B and or FST overexpressing conditions. Results: Applying Wald test we identified 423DEGs for FST+LIN28B(q-value< 0.01), 764 DEGs for LIN28B (q-value< 0.01) and 37 DEGs for FST (q-value< 0.05). The list of downregulated genes included structural genes (Tecta, Col2a1, Col11a2, Col9a3) as well as transcription factor known to promote neuronal/glial cell differentiation (Zbtb20,Nfix, Nfib) and cochlear maturation (Thrb).The list of upregulated genes contained a large number of let-7 target genes known to promote in stemness (e.g., Hmga2, Arid3a, Trim71,Igf2bp1), and to regulate stem cell metabolism (e.g.,Hk2),as well as genes involved in cell cycle regulation (e.g.,Ccnd2,Ccnd3, Cdkn1a, Cdkn2a) and cell fate specification (e.g. Sox11).
