Mgiseq platform

GSC11 (3.5×10⁵ cells/well) was seeded in a 6-well plate (laminin-coated). Cells were transfected with either shNT control or shMEIS1_72 lentivirus and then incubated for 48 h. At the end of the incubation period, cells were resuspended in TRIzol® Reagent (Invitrogen; CA, USA) and kept at −80°C. Libraries were constructed using the MGIEasy RNA Directional Library Prep Kit (MGI Tech., Shenzhen, China) following the manufacturer’s procedure. RNA quality control and high-throughput RNA sequencing were performed on the MGISEQ platform (MGI Tech) with 149 bp paired-end reads. Reads were mapped to the hg19 reference genome and expression values were calculated in fragments per kb transcript per million fragments mapped (FPKM). LAS Co., Ltd. (Gimpo, South Korea) performed the RNA purification, library preparation, and sequencing procedures. The new generation of high-throughput sequencing was used to study the differences between the two samples and gene expression in both groups was compared using EdgeR. Significant genes (Log2(FC) cut-off >0.5 and <0.5; p ≤ 0.05) were used for functional annotation on the DAVID website. GSEA analysis was also performed to identify the downregulated gene sets after MEIS1 knockdown.

Total RNA (1 µg) was processed to prepare the mRNA sequencing library following the manufacturer’s instructions provided with the MGIEasy RNA Directional Library Prep kit (#1000006386; MGI Tech, Shenzhen, China). The constructed library was quantified using a QauntiFluor^® ssDNA System (E3190; Promega). Subsequently, the prepared DNA nanoballs were sequenced on the MGISeq platform (MGI Tech) with 100 bp paired-end reads. FastQC (v0.11.9) was used to assess the read quality. Common sections of the MGISEQ adapter sequences were eliminated using TrimGalore (v0.6.5). The resulting trimmed reads were mapped to the GRCm38 (mm10) mouse reference genome using STAR (v2.7.3a) [24 (link)] with default configurations. To quantify gene expression levels, we used RSEM (v1.3.3) [25 (link)] along with the GRCm38.86 gene annotation to obtain the expected read counts and transcript per million (TPM) values.