GSM8290505: 3M AP 20dpl rep1; Mus musculus; RNA-Seq
To identify the lesion, 500 ul of 1% neutral red dye in PBS was injected intraperitoneally 2h prior to cardiac perfusion with ice-cold RNA-ase free 1X PBS. Spinal cords were flash frozen in isopentane and embedded in OCT on dry ice. Spinal cords were extracted and lesion and distant contralateral non-lesion tissue were microdissected out and flash frozen on dry ice. RNA was isolated using the Direct-zol RNA Miniprep Kits (Zymo Research #R2050) and sent to Azenta Life Sciences (South Plainfield, NJ, USA) for RNA quality check, library preparation, sequencing and analysis. RNA sequencing libraries were prepared using the NEBNext Ultra II RNA Library Prep for Illumina using manufacturer's instructions (NEB, Ipswich, MA, USA). Briefly, mRNAs were initially enriched with Oligod(T) beads. Enriched mRNAs were fragmented for 15 minutes at 94 °C. First strand and second strand cDNA were subsequently synthesized. cDNA fragments were end repaired and adenylated at 3'ends, and universal adapters were ligated to cDNA fragments, followed by index addition and library enrichment by PCR with limited cycles. The sequencing libraries were validated on the Agilent TapeStation (Agilent Technologies, Palo Alto, CA, USA), and quantified by using Qubit 3.0 Fluorometer (Invitrogen, Carlsbad, CA) as well as by quantitative PCR (KAPA Biosystems, Wilmington, MA, USA).