GSM9177255: Aganglionic colonic tissue specimen from HSCR patient 1; Colon; RNA-Seq
Total RNA was extracted from colonic tissue samples using TRIzol reagent kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol. RNA quality was assessed with an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA) and by RNase-free agarose gel electrophoresis. Eukaryotic mRNA was enriched using Oligo(dT) beads, fragmented into short fragments using fragmentation buffer, and reverse-transcribed into cDNA using the NEBNext Ultra RNA Library Prep Kit for Illumina (NEB #7530, New England Biolabs, Ipswich, MA, USA) with random hexamer primers. The resulting double-stranded cDNA was end-repaired, A-tailed, and ligated to Illumina sequencing adapters. Ligated products were purified using AMPure XP beads (1.0X) and amplified by PCR to generate the final cDNA libraries.