Organoid RNA Sequencing and Differential Expression Analysis

The organoid complex was removed from the hydrogel using the same method described above. Total RNA was extracted by adding TRIzol® reagent (Invitrogen, USA) to the multicellular precipitates. The whole-transcriptome sequencing library was constructed using the TruSeqTM RNA sample preparation Kit from Illumina (San Diego, CA). The paired-end RNA-seq sequencing library was sequenced on an Illumina NovaSeq 6000 sequencer with a read length of 2 × 150bp. The raw paired-end reads were trimmed and quality controlled using fastp (https://github.com/OpenGene/fastp)1 with default parameters. The clean reads were then aligned to the reference genome using HISAT2 (http://ccb.jhu.edu/software/hisat2/index.shtml)2 software in orientation mode. The mapped reads of each sample were assembled using StringTie (https://ccb.jhu.edu/software/stringtie/) in a reference-based approach3. The expression level of each gene was calculated using the transcripts per million reads (TPM) method in order to identify differentially expressed genes (DEGs) between different groups. Differential expression analysis was performed using DESeq24/DEGseq5/edgeR6/Limma7/NOIseq8. DEGs with |log2 (foldchange)| ≥ 1 and P-adjust ≤ 0.05 (DESeq2/edgeR/Limma) / P-adjust ≤ 0.001 (DEGseq) / Prob > 0.8 (NOIseq) were considered to be significantly differentially expressed genes.