Myocardial gene expression of type 2 diabetic mice with and without treatment with glucokinase activator
Myocardial gene expression of type 2 diabetic mice with and without treatment with glucokinase activator
Mice were terminally anaesthetized, hearts rapidly excised into ice cold PBS and freeze-clamped using wollenberger tongs. RNA extracted from the snap-frozen hearts was analysed by massive analysis of cDNA End (MACE-Seq, 1x75 bps Illumina; MACE: 5 Million reads / library). MACE-Seq was annotated, reads quantified and p values for differential expression generated by GenXPro (Frankfurt, Germany). Rapid MACE-seq was used to prepare 3â€™ RNA sequencing libraries. Samples of 100 ng of DNA-depleted RNA were used for library preparation, using the Rapid MACE-Seq kit (GenXPro GmbH, Germany). Fragmented RNA underwent reverse transcription using barcoded oligo(dT) primers containing TrueQuant unique molecular identifiers, followed by template switching. PCR amplified libraries were purified by solid phase reversible immobilization beads and subsequent sequencing was performed using the Illumina platform NextSeq 500. Unprocessed sequencing reads were adapter-trimmed and quality-trimmed using Cutadapt (version 3.4). Deduplication based on UMIs (Unique Molecular Identifier) was performed using in-house tools. FastQC (0.11.9) was used to assess the quality of sequencing reads. MultiQC (version 1.16) was used to create a single report visualising output from multiple tools across many samples, enabling global trends and biases to be quickly identified.