GSM7706572: Chem_GliaBAC_3; Mus musculus; RNA-Seq
For isolation of EGCs, the longitudinal smooth muscle layer and associated myenteric plexus were peeled off the wall of the smal intestineand subjected to enzymatic digestion to prepare a single-cell suspension. Single-cell suspensions were subjected to fluorescence activated cell sorting and live EGCs were isolated based on tdT expression DAPI exclusion. Cells were sorted directly into Trizol. Total RNA was isolated using PureLink RNA Micro Kit (Invitrogen #12183016) according to the manufacturer's instructions. Double stranded full-length cDNA was generated using the Ovation RNA-Seq System V2 (NuGen Technologies). Following quantification on a Qubit 3.0 fluorometer (Thermo Fisher Scientific), cDNA was fragmented to 200bp by acoustic shearing using Covaris E220 instrument (Covaris) at standard settings. The fragmented cDNA was then normalized to 100ng, which was used for sequencing library preparation using the Ovation Ultralow System V2 1-96 protocol (NuGen Technologies). A total of 7 PCR cycles were used for library amplification. The quality and quantity of the final libraries were assessed with TapeStation D1000 Assay (Agilent Technologies). The libraries were then normalized to 2,5 nM, pooled and loaded onto a HiSeq4000 (Illumina) to generate 75bp single-end reads.