RNA-seq of a small molecule targeted recruitment of a ribonuclease to degrade the c9ALS/FTD r(G4C2) repeat expansion

Published: 2 August 2021| Version 1 | DOI: 10.17632/332kvzz8xk.1
Contributors:
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Haruo Aikawa,
Yuquan Tong,
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,
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Description

We are publishing a study of a small molecule targeted recruitment of a ribonuclease to degrade the c9ALS/FTD r(G4C2) repeat expansion. We performed multiple RNA-seq experiments to evaluate the effect of the compound on transcriptome, including the 4 datasets: SET1. RNA-seq analysis of c9ALS patient-derived iPSCs treated with 50 nM of 7 plotted as average Log2(Fold Change) vs -Log10(q-value) (n = 1 C9orf72 iPSC line, 3 replicates per line). The amounts of mutant allele containing the repeat expansion were calculated using the coding SNPs as a proxy for abundance. SET2. RNA-seq analysis of iPSCs from healthy donors treated with 50 nM of 7 plotted as averege Log2(Fold Change) vs -Log10(q-value) (n = 1 C9orf72 iPSC line, 3 replicates per line). The amounts of mutant allele containing the repeat expansion were calculated using the coding SNPs as a proxy for abundance. SET3. RNA-seq analysis of +/+PWR500 mice treated with 33 nmol of 7 (n = 3 mice per treatment group). The amounts of mutant allele containing the repeat expansion were calculated using the coding SNPs as a proxy for abundance. SET4. RNA-seq analysis of c9ALS patient-derived iPSNs treated with 50 nM of 7 plotted as average Log10(TPM) (n = 1 C9orf72 iPSN line, 3 replicates per line). The amounts of mutant allele containing the repeat expansion were calculated using the coding SNPs as a proxy for abundance.

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Steps to reproduce

Patient-derived iPSCs were differentiated into motor neuron cells as described in our paper. At Day 21, the cells in Matrigel-coated 6-well plate were treated with 3 μM C9orf72-ASO, 3 μM Control-ASO and various concentrations of compounds. For the compound treatment, the medium was changed every 3 to 4 days with fresh compounds. After 2 weeks’ treatment, total RNA was extracted with Qiagen RNeasy Mini Kit. The cleavage of target G4C2-containing C9orf72 variants was confirmed by RT-qPCR with primers. The RNA samples were then subjected to RNA-seq analysis using Kallisto and Sleuth.