GSM9701785: DEL_MANF_2; Homo sapiens; RNA-Seq
Kidney organoids were enzymatically dissociated into single-cell suspensions using Accutase cell dissociation reagent (Gibco). Organoids were incubated in Accutase at 37 °C with gentle shaking for 5 minutes. Enzymatic digestion was terminated by adding an equal volume of DMEM/F12 (Gibco) supplemented with 10% fetal bovine serum (Gibco). Following digestion, cell suspensions passed through a 40 μm cell strainer (Alkali Scientific) to remove residual clumps, yielding a single-cell suspension suitable for magnetic bead-based cell sorting. UMOD-positive cells were isolated using a magnetic bead-based immunocapture approach. Biotinylated polyclonal sheep anti-human UMOD antibody (R&D Systems) was incubated with Dynabeads Biotin Binder magnetic beads (Invitrogen). For each preparation, 100 μL of Dynabeads were conjugated with 2 μg of anti-UMOD antibody by incubation at 4 °C for 2 hours with gentle rotation. After washing to remove unbound antibody, the conjugated beads were incubated with the single-cell suspension at 4 °C for 1 hour to allow binding to UMOD-expressing cells. Bead-bound UMOD-positive cells were isolated using a magnetic separation stand. The bead-cell complexes were retained against the magnet, and unbound (UMOD-negative) cells were removed through multiple PBS washes. The enriched UMOD-positive fraction was then collected and processed for downstream RNA extraction using RNeasy Micro Kit (Qiagen). Takara-Clontech SMARTer Ultra Low RNA