MSCs_Kidney_Transcriptome_06
Total RNA was extracted using TRIzol (Invitrogen) and quantified by NanoDrop ND - 1000. RNA integrity was verified with criteria: concentration > 50 ng/L, RIN > 7.0, OD260/280 > 1.8, and total RNA > 1 g. PolyA - containing mRNA was captured using Dynabeads Oligo (dT) 25. The captured mRNA was cleaved at 94 (5 - 7 min), reverse - transcribed to cDNA with SuperScript II Reverse Transcriptase, and used to synthesize U - labeled second - stranded DNAs. Ends were A - tailed, fragments were size - selected with magnetic beads, the second strand was digested with UDG enzyme, and PCR was performed to construct a 300 bp  50 bp library (PCR steps: 95 for 3 min, 8 cycles of 98 for 15 s, 60 for 15 s, 72 for 30 s, and 72 for 5 min). Paired - end sequencing (PE150) was carried out on Illumina Novaseq 6000. After sequencing, QC was done by analyzing base type distribution and base error probability. The mouse gene annotation file was used for contrast ratio analysis, the mouse genome for alignment, StringTie for read assembly and quantification. Positional distribution of aligned reads and coverage depth were analyzed, and samples were clustered with outliers removed.