title: GSM5513926: Sample 6_Grafted_cornea_pbscontrol; Mus musculus; RNA-Seq
library_construction_protocol: Two weeks after corneal transplantation, corneal grafts were excised and immediately submerged in the RNAlater solution (Ambion, Austin, TX, USA). Total RNA was isolated from five corneas each group by using a NucleoSpin RNA isolation kit (Macherey-Nagal GmbH, Duren, Germany). The cDNA sequencing libraries were prepared using NEBNext Poly(A) mRNA Magnetic Isolation Module and NEBNext Ultra II RNA Library Prep Kit for Illumina by following the manufacturer's recommendations (NEB, Ipswich, MA, USA). Briefly, enriched RNAs were fragmented for 15 minutes at 94 °C. First and second strand cDNA synthesis were subsequently performed. The universal adapter was ligated to cDNA fragments, followed by index addition and library enrichment with limited cycle PCR. Sequencing libraries were validated on the Agilent TapeStation System (Agilent Technologies, Palo Alto, CA, USA), and quantified by using Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA) as well as by quantitative PCR (Applied Biosystems, Carlsbad, CA, USA). The sequencing libraries were sequenced as 2x150 Paired End (PE) using the Illumina NovaSeq instrument according to manufacturer's instructions.