GSM8950947: Ileal organoid, L18-MDP, rep3; Mus musculus; RNA-Seq
To isolate single cells fo scRNA-seq, small intestine crypt-enriched tissue were washed in PBS prior to adding fresh prewarmed Trypsin (Corning, USA) and incubated at 37°C for 50 minutes, with mixing every 20 minutes. Tissues were filtered through a 40 mm mesh cell strainer on ice and pelleted by centrifugation at 4°C and washed twice with ice cold regular medium to remove debris. A manually estimated 4500 single cells per sample were then subjected to 10X Genomics single cell RNA sequencing following manufacturer's recommendations. For RNA-seq, RNA was isolated from ileal organoids using the RNeasy Mini Kit (QIAGEN), genomic DNA was removed using the TURBO DNase kit (Invitrogen). scRNA-seq libraries were prepared for sequencing using standard Illumina protocols. RNA-seq libraries were constructed using the stranded poly(A) mRNA library preparation kit (NEBNext).