GSM6449994: WT, EIPA, day 3, rep 2; Mus musculus; RNA-Seq
Organoid cultures were first washed with the cold (4°C) advanced DMEM/F12 (Invitrogen, 12634-028) supplemented with 10 mM HEPES (Invitrogen, 15630-080), 1 mM N-acetylcysteine (Sigma-Aldrich, A7250), 1X glutaMAX (Invitrogen, 35050-061), and 1X pen/strep. After the washing, wells containing the organoids were incubated with 300 uL cold (4°C) Corning recovery solution (Corning, 354253) for 15 min to melt the Matrigel. Next, organoids were mechanically disrupted with the P1000 pipette tips first and then trypsinized with RT TrypLE Express (Gibco, 1952062) for up to 15 min to disassociate into single cells. Single cells were then suspended within a sorting buffer that contained Hanks' balanced salt solution (HBSS) with 3% m/v FBS, 10 mM HEPES and 5 mM EDTA. After staining with Live/Dead (Invitrogen, L23105, 1:1000) in the sorting buffer, fluorescence-activated cell sorting (FACS) was performed on the BD FACSAira instrument to enrich either live cells (-/+ EIPA and -Dox conditions) or live GFP+ cells (+Dox condition). Around 100,000-150,000 cells per genotype/condition were collected and re-suspended in ~200 mL 1X PBS containing 0.04 % (w/v) BSA (Ambion, AM2616). Approximately 20,000 cells per genotype/condition were loaded onto a 10X Genomics Chromium Next GEM chip to generate a single cell 3' gene expression library using the 10X Chromium Next GEM Single Cell 3' regent kits v3.1. Pooled libraries were sequenced (single indexing) on a Illuminar NovaSeq 6000 sequencer with a standard sequencing protocol (Read 1, 28 cycles; i7 Index, 8 cycles; i5 Index, 0 cycles; Read 2, 91 cycles).