Corall total rna seq library preparation kit

In this study, 10 control samples, 10 paroxysmal AF, and 10 persistent AF samples were prepared for RNA sequencing. RNA was extracted from Approximately 500 mg AF and normal samples using the RNeasy mini kit (QIAGEN).
We next applied Corall Total RNA Seq library preparation kit (Lexogen, Vienna, Austria) for RNA Seq library using 150 ng of total RNA. The RiboCop rRNA Depletion Kit (Lexogen, Vienna, Austria) was used to remove rRNA. The quality of the sequencing library was evaluated by D1000 screen tape analysis using the 4200 TapeStation system (Agilent, United States) and quantified using QubitTM dsDNA HS analysis kit (Invitrogen, United States). RNA processing was used by Illumina NextSeq 500 sequencing. The R software package Deseq2 was used for mRNA differential expression analysis (Zhang et al., 2020 (link)). The genes with |log2 fold change | ≥ 1 and FDR ≤ 0.1 were considered to be differentially expressed (Gu et al., 2021a (link),b (link)).

Cells were harvested in PBS and pelleted. Pellets were resuspended in buffer A [5 mM Pipes (pH 8.0), 85 mM KCl, and 0.5% NP-40 substitute] and incubated on ice for 10 min. After centrifugation (5000 rpm for 5 min at 4°C), the supernatants containing the cytoplasm were removed and the nuclei-containing pellets were washed in buffer A without detergent. Nuclear RNA was extracted with TRIzol reagent (Thermo Fisher Scientific) according to the manufacturer’s instructions, using 5PRIME Phase Lock Gel heavy tubes (Quantabio) to separate the aqueous phase from the interphase/organic phase.
Sequencing libraries were generated using the RiboCop rRNA depletion kit followed by the CORALL Total RNA-Seq library preparation kit (both from Lexogen), following the manufacturer’s instructions and thereafter sequenced paired-end 50 bp with a NovaSeq 6000 (Illumina) at the Science for Life Laboratory National Genomics Infrastructure (Stockholm, Sweden).