GSM8864207: MC3_N18-23; Homo sapiens; RNA-Seq
Total RNA was isolated using the PureLink RNA Mini Kit after Trizol lysis (Thermo Fisher Scientific) from age-matched non-diabetic (n = 10) human corneas following the manufacturer's guidelines. The PureLink DNase Set (Thermo Fisher Scientific) was utilized for an on-column DNase digestion. Samples were eluted in RNase-free water. The concentration and quality of the RNA was evaluated using a NanoDrop 8000 spectrophotometer (Thermo Fisher Scientific), Qubit 2.0 Fluorometer (Thermo Fisher Scientific), and Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Total RNA samples were analyzed for RNA integrity on the 2100 Bioanalyzer using the Agilent RNA 6000 Nano Kit (Agilent Technologies, Santa Clara, CA) and quantified using the Qubit RNA HS Assay Kit (ThermoFisher Scientific, Waltham, MA). The SMART-Seq V4 Ultra Low RNA Input Kit for Sequencing (Takara Bio USA, Inc., Mountain View, CA) was used for reverse transcription and generation of double-stranded cDNA for subsequent library preparation using the Nextera XT Library Preparation Kit (Illumina, San Diego, CA). An input of up to 10 ng RNA was used for oligo(dT)-primed reverse transcription, followed by cDNA amplification and cleanup. Quantification of cDNA was performed using Qubit (Thermo Fisher Scientific). cDNA normalized to 80 pg/ml was fragmented, and sequencing primers were added simultaneously. A limiting-cycle PCR added Index 1 (i7) adapters, Index 2 (i5) adapters, and sequences required for cluster formation on the sequencing flow cell. Indexed libraries were then cleaned, library concentration was measured with a Qubit fluorometer (ThermoFisher Scientific), and library size was evaluated on a 4200 TapeStation (Agilent Technologies). Multiplexed libraries were sequenced on a NovaSeq 6000 (Illumina, San Diego, CA) using 75 bp single-end sequencing.
GEO
GSM8864207_r1