GSM9241653: Pdpn_WT_01; Mus musculus; RNA-Seq
Kidneys were minced and digested enzymatically (collagenase II, dispase, DNase I) to generate single-cell suspensions. Podoplanin-positive (PDPN⁺) cells were enriched by magnetic cell sorting (MACS) or fluorescence-activated cell sorting (FACS) using anti-PDPN antibody. Enriched cell pellets were lysed in TRIzol reagent (Invitrogen) and total RNA was isolated according to the manufacturer's instructions. RNA was further purified with the RNeasy Mini Kit (Qiagen), including on-column DNase digestion. RNA quality and integrity were assessed by Agilent Bioanalyzer, and only samples with RIN ≥ 8 were used for library construction. Total RNA (RIN ≥ 8) from Pdpn⁺ cells was poly(A)-enriched using oligo-dT magnetic beads (e.g., NEBNext Poly(A) mRNA Magnetic Isolation Module or equivalent). Stranded Illumina libraries were prepared with the NEBNext Ultra II Directional RNA Library Prep Kit following the manufacturer's instructions: RNA fragmentation, first- and second-strand cDNA synthesis (dUTP method for strand specificity), end repair/A-tailing, adaptor ligation, and limited-cycle PCR enrichment with unique dual indexes. Libraries were purified with SPRI beads, quantified by Qubit, and fragment size distributions were verified on an Agilent Bioanalyzer. Equimolar pools were sequenced on an Illumina NovaSeq 6000 (paired-end, 2×150 bp).