Novaseq system

RNA of Gltscr1^−/− and Gltscr1^+/+ cardiac tissues collected from E12.5, E13.5, and E15 embryos were extracted, sequenced, and analyzed by bioacme (Wuhan, China). Each group collected three embryos from the same parent and were used for condition. The cDNA libraries were prepared from high-quality RNA using an Illumina TruSeq RNA sample prep kit following the manufacturer’s instructions (Illumina, San Diego, CA, USA). The individual RNA-seq libraries were pooled based on their respective sample-specific 6-bp adaptors and sequenced at 150-bp/sequence pair-read using an Illumina NovaSeq system. Clean Reads were mapping into the hg19 reference genome by STAR and quantified by RSEM. Differential expression genes were identified by DESeq2 in R. Benjamini–Hochberg false discovery rate method was applied to correct for multiple hypothesis testing. Genes with P < 0.05, fold change > 1.5, or fold change < 0.67 were defined as different expression genes as candidates for further analysis. Gene expression Heatmap was accomplished with R package pheatmap. Gene Ontology enrichment analysis was performed using DAVID. The results were visualized by the R package ggplot2 in R software. All raw and processed sequencing data generated in this study have been submitted to the NCBI Sequence Read Archive (SRA, https://www.ncbi.nlm.nih.gov/sra) under accession number PRJNA820129.

RNA-seq libraries were performed as previously described (He et al., 2018 (link)). 200 ng of total RNA calculated by Qubit 2.0 (Invitrogen) was prepared for library construction. RNA-seq libraries were generated using the KAPA Stranded mRNA-Seq kit according to the manufacturer’s manual. In Short, the mRNA was enriched with oligo magnetic beads. Then cDNA was synthesized using random hexamer primers and purified with 1.0× Agencourt AMPure XP beads (Beckman). Finally, the cDNA fragments (approximately 300 bp) linked with sequencing primers were isolated by gel electrophoresis and amplified by PCR. The sequencing was performed by Berry Genomics Co Ltd. using the NovaSeq system developed by Illumina. 2 or 3 biological replicates were analyzed for each treatment condition.

Metagenomic DNA extracts as well as sorted single cell MDA products were quantified using the Qubit dsDNA HS Assay Kit (ThermoFisher Scientific, OR, United States). Illumina Sequencing libraries were then prepared using the NEBNext^® Ultra^TM DNA Library Prep Kit and NEBNext^® UltraTM II FS DNA Library Prep Kit (New England BioLabs, Frankfurt, Germany), respectively, using unique dual index adapters and following the manufacturer’s instruction. Resulting library fragment lengths were assessed using the Agilent 2100 Bioanalyzer with a High Sensitivity DNA Kit (Agilent Technologies, Germany). The libraries were then pooled and sequenced on an Illumina NovaSeq system using a paired-end approach with 150 cycles per read. Sequencing depths of approximately 6.8 million read pairs or 2 Gb were produced per SAG, on average.