GSM9156049: P7Ascl1_cKO single cell Tomato-; Mus musculus; RNA-Seq
long and small RNA: Total RNA was isolated using the RNeasy Plus Mini Kit (Qiagen). The SMART-Seq HT Kit (Takara) was utilized to generate complementary DNA (cDNA) from 1 ng of total RNA input. The synthesis process included 13 PCR cycles. Single-cell RNA sequencing was performed using pre-templated instant partitions (PIP) sequencing (PIPseq, (Lee et al., 2024)). single cell RNA-seq: The T20 kit (≤ 20,000 cells output, FBS-SCR-T20-4-V4.05, Fluent Biosciences) was used according to the manufacturer's instructions. In brief, tubes containing template beads were thawed on ice, and the required number of input cells was added per tube (i.e., approximately 25,000–30,000 live cells fora split T20 kit, 2x "T10"). The suspension was mixed gently by pipetting and processed further according to the manufacturer's instructions. After the cDNA synthesis, purification, and library preparation, the concentration, integrity, and quality were assessed using the Agilent 2100 Bioanalyzer System and the high-sensitivity DNA kit (Agilent Technologies, Santa Clara, CA, USA). For bulk long RNA library preparation, cDNA was quantified using the Invitrogen Quant-iT system and subsequently diluted to a concentration of 0.3 ng/μL for library preparation. Libraries were prepared using the Nextera XT DNA Library Preparation Kit, employing a total of 12 PCR cycles to amplify and uniquely barcode the samples. Small RNA libraries were prepared from 10 ng of purified miRNA using the SMARTer® smRNA-Seq Kit for Illumina (Takara Bio), following the manufacturer's protocol. The kit utilizes a ligation-free method based on SMART (Switching Mechanism at the 5′ end of RNA Template) technology to generate high-quality libraries from low-input small RNA. After adapter ligation and cDNA synthesis, libraries were amplified using PCR and purified using AMPure XP beads according to the protocol specifications. The quality and size distribution of the final libraries were assessed using the Agilent High Sensitivity D1000 ScreenTape on the TapeStation 4200 system (Agilent Technologies) to confirm the presence of expected small RNA insert sizes (~140–150 bp for miRNA). Library concentrations were measured using the Quant-iT™ dsDNA High Sensitivity Assay Kit (Invitrogen) to ensure accurate quantification. Libraries were normalized to equimolar concentrations based on fluorometric measurements and pooled accordingly. Sequencing was performed on the Illumina NovaSeq X+ platform using paired-end 100-cycle reads, providing high-depth coverage suitable for small RNA transcriptome analysis. Small RNA libraries were prepared from 10 ng of purified miRNA using the SMARTer® smRNA-Seq Kit for Illumina (Takara Bio), following the manufacturer's protocol. The kit utilizes a ligation-free method based on SMART (Switching Mechanism at the 5′ end of RNA Template) technology to generate high-quality libraries from low-input small RNA. After adapter ligation and cDNA synthesis, libraries were amplified using PCR and purified using AMPure XP beads according to the protocol specifications. The quality and size distribution of the final libraries were assessed using the Agilent High Sensitivity D1000 ScreenTape on the TapeStation 4200 system (Agilent Technologies) to confirm the presence of expected small RNA insert sizes (~140–150 bp for miRNA). Library concentrations were measured using the Quant-iT™ dsDNA High Sensitivity Assay Kit (Invitrogen) to ensure accurate quantification. Libraries were normalized to equimolar concentrations based on fluorometric measurements and pooled accordingly. Sequencing was performed on the Illumina NovaSeq X+ platform using paired-end 100-cycle reads, providing high-depth coverage suitable for small RNA transcriptome analysis.  For scRNA-seq, after the cDNA synthesis, purification, and library preparation, the concentration, integrity, and quality were assessed using the Agilent 2100 Bioanalyzer System and the high-sensitivity DNA kit (Agilent Technologies, Santa Clara, CA, USA). For scRNA-seq, after the cDNA synthesis, purification, and library preparation were conducted following the manufacturer's instructions. The concentration, integrity, and quality were assessed using the Agilent 2100 Bioanalyzer System and the high-sensitivity DNA kit (Agilent Technologies, Santa Clara, CA, USA).