RNA-seq Analysis of TBXT Knockdown in hESCs

Total RNA was harvested from day 3 NMPs obtained from TBXT shRNA sOPTiKD hESC in the presence and absence of Tet (three biological replicates) using the total RNA purification plus kit (Norgen BioTek) according to the manufacturer’s instructions. Sample quality control, library preparation, and sequencing were carried out by Novogene (http://en.novogene.com). Library construction was carried out using the NEB Next Ultra RNA Library Prep Kit and sequencing was performed using the Illumina NoveSeq platform (PE150). Raw reads were processed through FastQC v0.11.2 and Trim Galore. Reads were aligned using STAR v2.4.2a (Dobin et al., 2013 (link)) to the human reference genome assembly GRCh38 (Ensembl Build 79) in the two-pass mode. The RSEM v1.3.0 (Li and Dewey, 2011 (link)) was used to extract expected gene counts, where genes expressed in <3 samples or with total counts ≤5 among all samples were excluded. We identified genes showing significant differential expression with DESeq2 (Love et al., 2014 (link)), with log2FoldChange>|0.5| and Benjamini–Hochberg-adjusted p<0.05. Data were deposited to GEO (Accession number: GSE184620).