GSM7430627: XMOOl WT DAY 100, scRNAseq; Homo sapiens; RNA-Seq
Five to six 100 day old organoids were collected and placed in a 35 mm plate. To remove dead cells from the organoid core, organoids were cut in half using a syringe needle and washed with PBS. Subsequently, PBS was removed and 200 ul of TrypLE Express solution (12604-013 Gibco) was added. Next, the organoids were cut into smaller fragments using two needles attached to 1 ml syringes. Fragments were collected in a 1.5 ml reaction tube and subjected to gentle pipetting followed by intermittent resting at 37C for 5 minutes. Single cells present in the supernatant after 5 min were collected into a new reaction tube and placed into the incubator. Tissue fragments remaining in the tube were treated with 100 ul of Triple Express and dispersed into small pieces using consecutive pipetting with 1 ml and 200 µl pipette tips. After additional five minutes, the single cells in the supernatant were again collected and added to the tube containing the single-cell suspension. This procedure was repeated 3 time for a total of 15 min. Any remaining tissue fragments were discarded. Triple Express dissociation was stopped by adding an equal volume of E8 Stem Flex medium, resulting in a total volume of 1 ml. Cells viability was assessed using cell viability dye Trypan blue (0.4%, T10282, Invitrogen) and cells were counted using an automated cell counter (Countess II, Invitrogen). Thereafter, the single-cell suspension was filtered through a 40 um filter (22363547, Thermo fisher) placed on top of a 50 ml tube. Filtered cells were collected and cell viability determined as above. Typically, cell viability at this step was around 50%-60%. To increase the precent of live cells in the single-cell suspension, the cells were centrifuged for 2 min at 200g and placed on ice. The cell pellet was washed in 1 ml of PBS with 0.04% BSA (130-091-376, Miltenyibiotec), pipetted gently to remove any cell clumps, and counted again. This washing step was repeated two more times, resulting in a cell viability of approximately 80%. Finally, 5000 cells were collected at a concentration of 1000 cells/ul in PBS, 0.04% BSA for library preparation and sequencing. Libraries were generated using the Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 Dual Index (10X Genomics Inc., USA). Briefly, a droplet emulsion targeting 5,000 cells was generated in a microfluidic Next GEM Chip G, followed by barcoded cDNA generation inside the droplets. Purified and amplified cDNA was then subjected to library preparation and sequenced on a NovaSeq 6000 instrument (Illumina, USA) using a SP100 flow cell kit to a depth of 400M read-pairs per sample. Sequencing was performed as a service at the Department of Molecular Medicine (MOMA) of Aarhus University Hospital, Denmark (https://www.moma.dk/).