RNA-seq libraries were prepared from total RNAs from the collected neuronal cells of 12 samples (three biological replicates, i.e., independent cell cultures, of four distinct groups). These sample groups are for TCF4 expression knockdown and control at hiPSC-derived NPC stage (day 3 after differentiation) and Glut_N stage (day 14 after differentiation). Total RNAs were extracted as described above. Three main methods for quality control (QC) of RNA samples were conducted, including (i) preliminary quantification, (ii) testing RNA degradation and potential contamination (Agarose Gel Electrophoresis), and (iii) checking RNA integrity and quantification (Agilent 2100) (10). After the QC procedures, a library was constructed and library QC was conducted consisting of three steps, including (i) testing the library concentration (Qubit 2.0), (ii) testing the insert size (Agilent 2100), and (iii) precise quantification of library effective concentration (qPCR) (10). The quantified libraries were then sequenced using Illumina HiSeq sequencers with 150-bp paired-end reads, after pooling according to its effective concentration and expected data volume.

The paired-end sequenced reads for each sample were obtained as FASTQ files. Initial quality check of the raw data was performed using FastQC. All FASTQ files passed the QC stage, including removing low-quality bases, adapters, short sequences, and checking for rRNA contamination. The average number of preprocessed reads per sample was ~22,740,000. High-quality reads were mapped to the reference genome GRCh38 using STAR (59). Sorting and counting were also performed using STAR based on the procedure recommended by the developers. To detect DE genes, the output read counts were fed to edgeR software package (60). Normalization, batch correction, and differential expression analysis were conducted using the recommended settings.

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