GSM8193394: mg_screen1_ascl1_oe; Mus musculus; RNA-Seq
In a 48-well plate, the cells were  rinsed with 200ul HBSS and then 200ul of Accutase. Accutase was removed, and the cells were incubated at 37°C for 5 minutes. Cells were then resuspended in 200 uL of 1% tet-FBS-reduced growth medium with a wide-bore P200 pipette tip to isolate single cells. The library was prepared by the original sci-RNA-seq2 protocol  (Cao et al., 2017) in combination with hashing as presented by sci-Plex (Srivatsan et al., 2019). Nuclei were fixed in the presence of a barcoded polyA DNA oligo. A reverse transcription reaction with barcoded RT primers captured 3'-polyAdenylated transcripts and the barcoded oligo. Using FACs, nuclei were sorted into wells of 96-well plates where second strand synthesis was followed by tagmentation of dsDNA. PCR was performed with barcoded primers and the library was size-selected. The unique combination of barcodes in the RT and PCR steps enable the reconstruction of individual cells in silico.