Bone marrow derived mesenchymal stem cell of homo sapiens: RNA Seq
RNA was extracted from cells in TRIzol using the RNeasy Mini Kit (Qiagen, Cat# 74104). For quality control (QC), all direct RNA samples were quantified using the Qubit RNA BR Assay (Invitrogen, Cat# Q10211). RNA purity was assessed with the QIAxpert, and RNA integrity was evaluated on the TapeStation using RNA ScreenTapes (Agilent, Cat# 5067-5576). Only QC-passed samples were taken forward for library preparation and sequencing. Libraries for mRNA sequencing were prepared using the KAPA RNA HyperPrep Kit (Roche KAPA, Cat# 0000141759). An initial input of 200 ng total RNA was used. mRNA molecules were captured using magnetic oligo(dT) beads (Roche KAPA, Cat# 7962240001). After purification, the enriched mRNA was fragmented using divalent cations at elevated temperatures (94C for 6 minutes). First-strand cDNA synthesis was performed using reverse transcriptase. Second-strand cDNA synthesis and A-tailing were carried out in a combined reaction, converting the cDNA:RNA hybrid to double-stranded cDNA (dscDNA), incorporating dUTP into the second strand, and adding dAMP to the 3 ends of the resulting dscDNA. Adapter ligation followed, using dsDNA indexing adapters with 3 dTMP overhangs. The adapter-ligated products were purified and enriched using high-fidelity, low-bias PCR. The strand containing dUTP was not amplified, ensuring strand-specific sequencing. PCR amplification was performed under the following thermal conditions: initial denaturation at 98C for 45 seconds; 13 cycles of 98C for 15 seconds, 60C for 30 seconds, and 72C for 30 seconds; followed by a final extension at 72C for 1 minute. PCR products were then purified and checked for fragment size distribution using the Fragment Analyzer with the HS NGS Fragment Kit (16000 bp) (Agilent, Cat# DNF-474-1000). Prepared libraries were quantified using the Qubit HS Assay (Invitrogen, Cat# Q32854). Libraries were then pooled and diluted to the final optimal loading concentration and loaded onto the Illumina NovaSeqX Plus instrument to generate 60.00 million, 150 bp paired-end reads per sample.