Illumina NovaSeq 6000 paired end sequencing; Effect of mycophenolic acid on MPC-1 murine podocytes
Effect of mycophenolic acid on MPC-1 murine podocytes
The podocyte cell line we used is a conditionally immortalized murine podocyte cell line that we originally received from the Mundel lab (http://dx.doi.org/10.1006/excr.1997.3739, https://doi.org/10.1038/sj.ki.5002291) and was cultivated on type I collagen (A1064401 – Invitrogen) coated culture dishes and kept in RPMI-1640 Medium + GlutaMAX (#61870036, Gibco), supplemented with 10% fetal bovine serum (FBS, 10270106 – Gibco), 1% HEPES solution (H0887 – Sigma) and 1% sodium pyruvate (S8636 – Sigma) at 33 °C in the presence of recombinant mouse Interferon- (INF-γ, #315-05, PeproTech) to allow proliferation at permissive conditions. This was followed by incubation at 37 °C without additional INF-γ for 10 days to induce differentiation at the non-permissive condition. After differentiation, cells were treated with Mycophenolic Acid (M3536 – Sigma-Aldrich) with a concentration of 10 mg/l for 2 hours, followed by a concentration of 4 mg/l for 22 hours (M). Cells of the control group received the respective amount of methanol as vehicle. Total RNA extraction was performed with the direct-zol RNA Miniprep kit (Zymo Research) following the manufacture's instructions. Library preparation and sequencing was performed by the Cologne Center for Genomics. Libraries were prepared using the Illumina TruSeq mRNA stranded sample preparation Kit. Library preparation started with 300 ng total RNA. After poly-A selection, mRNA was purified and fragmented using divalent cations under elevated temperature. The RNA fragments underwent reverse transcription using random primers, followed by second strand cDNA synthesis with DNA Polymerase I and RNase H. After end repair and A-tailing, indexing adapters were ligated. The products were then purified and amplified to create the final cDNA libraries. After library validation and quantification (TapStation 4200, Agilent), equimolar amounts of library were pooled and quantified by using the Peqlab KAPA Library Quantification Kit and the Applied Biosystems 7900HT Sequence Detection System.