GSM8486327: ischemic reperfusion rep1; Mus musculus; RNA-Seq
Total RNA was isolated from the samples using Trizol reagents (Invitrogen, 15596018CN), where infarct core and peripheral regions were used in the MI and MISham groups, and infarct core tissue was used in the IR and IRSham groups. The SQK-PCB109 (Oxford Nanopore Technologies) kit was used to prepare a full-length transcriptome sequencing library. In detail, 2 μM VN Primer (VNP) and 10 mM dNTPs were added and incubated at 65° C for 5 min. Then snap cooled the samples and add 5xRT Buffer, RNaseOUT, and 10 μM Strand-Switching Primer (SSP) at 42℃ for 2 min. Next, 1 μl of Maxima H Minus Reverse Transcriptase was added, which allowed the full-length transcript to be reversely transcribed into cDNA in vitro for further PCR. Sequencing uses PromethION (FLO-PRO002) to generate fast5 files and the sequencing was scheduled to run for 72 hr or stop early when there are less than 10 active pores. Reads with quality score (Q score) < 7 were filtered out to ensure the quality of sequencing. Next-generation sequencing (Illumina)-based RNAseq was applied to the same samples. In total, 16 samples were sequenced for four groups (MI, IR, MI-Sham, and IR-Sham) with four samples in each group.