GSM9657935: clone10_1; Mus musculus; RNA-Seq
Organoids with MADM clones induced at D13 were collected at D20 for MADM-CloneSeq. The sample collection steps for MADM-CloneSeq were adapted from previously published protocols (PMID: 38968076). Organoids were kept in cell culture membrane (Millicell) in oxygenated (95% O2, 5% CO2) artificial cerebrospinal fluid (ACSF) containing (in mM): 118 NaCl, 2.5 KCl, 1.25 NaH2PO4, 1.5 MgSO4, 1 CaCl2, 10 Glucose, 3 Myo-inositol, 30 Sucrose, 30 NaHCO3 prepared   with ultra-distilled water; pH=7.4, at 35C, until sample collection. All work surface and equipment were cleaned using RNase away solution (Thermo Fisher Scientific) prior to experiments. Single organoids were transferred to a LNscope 240XY miscroscope (Luigs & Neumann) and superfused with ACSF at a rate of 0.5 ml/min at room temperature. Individual clones were visualized under a 20x objective and by using ImageJ software. To observe red or green fluorescence, light was emitted from a LED light source (CoolLED, Andover, UK). Glass pipettes (B150-86-10, Sutter Instrument, Novato, CA) previously autoclaved, were pulled using a P-1000 pipette puller (Sutter Instrument) to generate pipettes with an opening of around 3-5ym. Before use, each pipette was filled with 3.5yl of pipette solution which consisted of RNase inhibitor (Takara, 1.95%) in RNase-free PBS (Invitrogen) filtered through a 0.22mm filter. Red or green fluorescent neurons were approached with the pipette under positive pressure to avoid tissue contamination of the pipette tip. When contact with cell membrane was made, tight seal formation was achieved under IR/DIC visualization. The complete aspiration of the cell body was immediately performed by applying a gentle and steady suction while monitoring under fluorescence and typically takes 5-10 s. The negative pressure was removed immediately once the target cell was collected and the pipette was carefully withdrawn from the acute slice to avoid contamination. The content of each pipette was directly transferred into individual PCR tubes and kept at -80C until cDNA library preparation. cDNA libraries for individual MADM-CloneSeq cells were were prepared using Smart-seq3 protocol (PMID 32518404) in 3 batches (96well plates).