GSM6630338: OEC-1; Rattus norvegicus; RNA-Seq
Flasks with purified OECs and leftover cells were rinsed with HBSS and dissociated with trypsin-EDTA as above. Cells were washed in D/F-FBS-P/S medium and further resuspended with silconized pipettes. Media and cells were washed and centrifuged at 365 g for 10 min, and resuspended. After the final wash, cells for sequencing were resuspended in PBS+BSA in volumes of 1200 cells/µL. 4 flasks of cells (2 purified OECs and 2 'leftovers') were generated on each of 2 different culture dates for scRNA-seq, yielding 8 samples in total (4 purified OECs and 4 'leftovers'). Extra cells from the same preparation were plated on PLL-coated coverslips in 24-well plates with 500µL D/F-FBS-P/S medium changed after 2 days. Cells were cultured for 3 days, fixed in cold 4% paraformaldehyde for 20 min, washed 3X with PB and stored in PB with 0.06% sodium azide. scRNA-seq was performed using the 10X Genomics Chromium scRNA-seq system which incorporates microfluidic capture-based encapsulation, barcoding, and library preparation (10X Genomics, Pleasanton, CA). About 10,000 cells per OEC or leftover preparation were loaded into a Chromium Chip B along with partitioning oil, reverse transcription reagents, and a mix of hydrogel beads containing 3,500,000 unique 10X barcodes.