GSM9594605: FLASH-seq Plate 1, 384 single cells; Mus musculus; RNA-Seq
Cells were trypsinized, washed with PBS, resuspended in FACS buffer (PBS, 0.5% BSA, 2-5 mM EDTA) at 1e6 cells/mL, and filtered through a 40 um mesh. DAPI was added to exclude dead cells. Single cells were sorted on a FACS S8 Discovery system into 384-well plates containing lysis buffer using a binned strategy (4 bins on Nr1d1-Venus, 2 bins on H2B-iRFP). RNA was denatured at 72C prior to reverse transcription. Full-length scRNA-seq libraries were generated using Mercurius FLASH-seq kits (Alithea Genomics). cDNA was amplified by PCR (21 cycles), quantified using PicoGreen dsDNA Assay (Thermo Fisher), diluted to 200 pg/uL, and tagmented. Sequencing adapters were added via secondary PCR (8 cycles). Libraries were sequenced on an Element AVITI system (Element Biosciences, CloudBreak) with paired-end 75 bp reads.