RNA-Seq profiling of human embryonic stem cells carrying different levels of mtDNA heteroplasmy cultured in mTeSR Plus and E8 media
RNA-Seq profiling of human embryonic stem cells carrying different levels of mtDNA heteroplasmy cultured in mTeSR Plus and E8 media
Cells were grown to 70-80% confluency, washed twice with PBS, and dissociated using ReleSR. Cell pellets were collected by centrifugation at 300 × g for 5 minutes at 4°C, snap-frozen in dry ice, and stored at -80°C until RNA extraction. For each biological replicate, an independent passage of cells was harvested. Human embryonic stem cells (H1 / WA01) carrying different levels of mtND6 mutations (wild-type, 5%, 56%, and 77%) were generated by mitochondrial base editing using DdCBE (Mok et al., 2020). Cells were maintained on Geltrex-coated plates (Thermo Fisher Scientific) in either mTeSR+ medium (STEMCELL Technologies) or in-house prepared Essential 8 (E8) medium at 37°C in a humidified incubator with 5% CO2. Medium was changed daily, and cells were passaged every 4-5 days using ReleSR. Cells were harvested at approximately 70-80% confluency for RNA extraction. Total RNA was extracted from cell pellets using the RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions. RNA concentration and purity were assessed using a NanoDrop spectrophotometer (Thermo Fisher Scientific). RNA samples were then shipped to Azenta Life Sciences for quality assessment and library preparation. RNA-seq libraries were prepared by Azenta Life Sciences using a poly(A) selection-based mRNA library preparation workflow. Briefly, mRNA was enriched from total RNA by poly(A) selection, fragmented, and reverse transcribed using random hexamer priming. First and second strand cDNA were synthesized, followed by end repair, 5' phosphorylation, and dA-tailing. Sequencing adapters were ligated, and libraries were PCR-enriched. Library quality and quantity were assessed prior to sequencing.