GSM5015811: Murine intestinal organoids - Dome-rep1; Mus musculus; RNA-Seq
Organoids were grown in either sandwich or dome culture in glass-bottom 12 well plates (Mattek;  P12G-1.5-14-F). Before dissociation, all pipette tips and transfer vessels were coated with 10% w/v BSA to  prevent loss of material. Media was aspirated and culture wells were washed twice with 1X PBS before  incubating in 1mL TrypLE Express (ThermoFisher Cat No. 12604-021). Upon addition of TrypLE Express,  a 1 mL pipette tip was used to mechanically detach organoids embedded in Matrigel from either sandwich  or dome cultures, taking care to scrape the bottom of the well with such force so as not to detach the glass  bottom coverslip, but to visibly remove the Matrigel from the coverslip. Organoids were incubated at 37°C  for between 20-30 minutes, monitoring every 5 minutes to check on the status of dissociation, and to  repeat brief mechanical disruption by trituration. Multiple wells (between 2 and 10) per condition were  pooled into a 15 mL falcon tube and diluted 1:1 with ice cold DMEM, and strained (40 μm [pluriSelect  43-10040-60]) into a 15 mL falcon tube. This cell solution was then centrifuged at 250xg in a spinning  bucket centrifuge for 1 minute at 4°C, and the suspension was aspirated. Cells were resuspended in 200  μL of 1% w/v BSA in 1X PBS, kept on ice, and cell count and viability were measured with a  hemocytometer immediately prior to single cell encapsulation with the inDrops method. InDrops single cell RNA sequencing was performed as described before [Zilionis et al., 2019, Immunity, 50, 1317-1334.e10; Zilionis et al., 2017, Nat Protoc, 12, 44-73; Klein et al., 2015, Cell, 161, 1187-1201] with changes made as written in the publication associated with this GEO record. All reads were processed with the indrops.py pipeline found at https://github.com/AllonKleinLab/klunctions/tree/master/sam/inDrops_pipeline using the parameters included in the provided bash script files: submit_indrops_sandwich_dome_rc.sh and submit_indrops_perturb2.sh, for sandwich v. dome and perturbation experiments, respectively. scRNA-seq