GSM8513191: E17.5; Mus musculus; RNA-Seq
Small intestines were excised from E15.5, E17.5 embryos or P7 mice, cut longitudinally in ice-cold PBS to remove the contents. The epithelium was separated from the mesenchyme by gentle shaking, followed by 70 μm cell sieve filtration.  The remaining mesenchymal flakes were prepared as described above for the separation of MCs from the P14 intestine to obtain a single-cell suspension. The resulting fractions of both mesenchymal and epithelial cells were combined and filtered through a 40 μm cell sieve, then incubated with antibodies on ice for 15 minutes in PBS containing 2 % FBS. The primary antibodies employed were anti-CD31-APC (eBiosciences, 17-0311-82, 1: 500), anti-CD326 (Epcam)-PeCy7 (eBiosciences, 25-9326-42, 1: 500) and anti-CD45-APC (eBiosciences, 17-0451-82, 1: 500). Fixable Viability Dye eFluor™ 450 (eBiosciences, 65-0863-14, 1: 1000) was applied to identify dead cells. After several washes in ice-cold PBS, cells sorting was performed on BD FACS Arial 3.0 machin. The 450-/Epcam+ and 450-/Epcam-/CD31-/CD45- cells were sorted into EP tubes in single-cell mode by FACS. Chromium Single Cell 3ʹ v2 libraries were sequenced with a Novaseq 6000 sequencer (Illumina), with the following sequencing parameters: read 1, 150 cycles; i7 index, 8 cycles and read 2, 150 cycles.