Novaseq6000 sequencing instrument

RNA sequencing and gene expression profiles were performed to determine the maturation of hiPSC-CMs in isolated culture versus BCP. Isolated total RNA was isolated from cultured hiPSC-CMs and BCP samples using a RNeasy Blood and Tissue kit (Qiagen, Carlsbad, CA, USA). To construct the sequencing library for Nova Seq 6000, 2 μg of total RNA was used for library construction with the Illumina TruSeq Stranded total RNA Library Prep Kit (cat # 20020596, CA, USA). Next, paired-end sequencing was performed using the Illumina Nova Seq 6000 sequencing instrument with NovaSeq 6000 S2 reagent kit (cat # 20012860, CA, USA), according to the manufacturer’s instructions, yielding 150-bp paired-end reads. EdgeR software was used for differential expression analysis of RNA sequencing and gene expression profiles with three biological replications. This analysis assumed a negative binomial distribution for statistics. To eliminate biological variation, the screening of differential genes needed to be evaluated in terms of difference multiples and significance levels. The screening threshold for differential genes in this analysis was set to |log2 (Fold Change) | > 1, FDR < 0.01.

The genomic DNA of feces was extracted using a QIAamp DNA Stool Mini Kit (Tiangen, Beijing, China) according to the manufacturer’s instructions. The integrity and size of the DNA were verified through 1% agarose gel electrophoresis, and DNA concentrations were determined. The 16S rRNA-based amplification process was executed by utilizing the primers 341F (5′-CCTAYGGGRBGCASCAG-3′) and 806R (5′-GGACTACNNGGGTATCTAAT-3′), which are directionally targeting the V3-V4 hypervariable regions of the 16S rRNA gene. The amplicons underwent a purification, quantification, and normalization process before being combined in equal molar concentrations. The sequencing of the pooled amplicons was then conducted using an Illumina Novaseq 6000 sequencing instrument (Illumina, Inc., San Diego, CA, USA).
Effective sequencing data were then clustered into operational taxonomic units (OTUs) with 97% similarity cut-off using USEARCH (version 11). The RDP Classifier (version 2.13) algorithm was used to annotate taxonomic information for each representative sequence using the Silva Database (SSU138). The Mothur program (version 1.30.2) was employed to analyze rarefaction curves and calculate richness estimators and diversity indices. Linear discriminant analysis (LDA) coupled with effect size (LEfSe) was applied to evaluate the differentially abundant taxon.

RNA libraries and next-generation sequencing (NGS) were conducted by Novogene Co., LTD (California, USA). For mRNA transcriptome profiling, RNA libraries were generated using a NEBNext^® Ultra™ RNA Library Prep Kit for Illumina (NEB E7530L) and for miRNA profiling, small-RNA libraries were prepared using a TruSeq Small RNA Library Prep Kit (Illumina RS-200) according to the manufacturer’s instructions. Library quantity and quality assessments were performed using a Qubit^® DNA HS Assay Kit on a Qubit^® 2.0 Fluorometer and an Agilent DNA High Sensitivity Kit in an Agilent 2100 Bioanalyzer, respectively. Library concentrations were calculated using quantitative PCR measures then pooled in equimolar ratios and sequenced in a NovaSeq6000 sequencing instrument (Illumina) as paired (150 bases) or single-end (50 bases) reads for the transcriptome and miRNA sequencing, respectively.