RNA sequencing (RNA-seq)

Total RNA was isolated from the brain tissues of five NTD embryos and five control embryos using TRIzol reagent (Invitrogen), following RNA concentration detection using the NanoDrop 2000. Ribosomal RNA (rRNA) was removed, and the RNA was fragmented. The RNA library was established using the Illumina TruSeq RNA Sample Prep Kit and then loaded on an Illumina HiSeq 2,500 platform for 150 bp paired-end sequencing. The raw data have been deposited in the NCBI Sequence Read Archive (SRA) database under accession number PRJNA879256.

Raw reads were subjected to data filtration to remove low-quality reads. Read alignment to the reference genome (Mus_musculus.GRCm39) was conducted using HISAT2 (v2.1.0) (Kim et al., 2015). lncRNA and mRNA read counts were generated using RSEM (Li and Dewey, 2011), and mRNA or lncRNA expression levels were quantified.

To explore the key molecules in the pathogenesis of NTD, differentially expressed genes (DEGs) and differentially expressed lncRNAs (DElncRNAs) between NTD and control samples were identified using the DESeq2 package (v1.22.2) in R based on RNA-seq data. The p-value was adjusted using the Benjamini–Hochberg (BH) method (Benjamini and Hochberg, 1995). The cutoff values were adjusted p < 0.05 and |fold change| > 1.