Nebnext ultra 2 rna library preparation kit

mRNA-seq and data analyses were performed as previously described [15 (link)]. Briefly, mRNA from total RNA was purified using poly T oligo-attached magnetic beads, and mRNA libraries were prepared using the NEBNext Ultra II RNA library preparation kit for Illumina (NEB), according to the manufacturer’s protocol. Qubit and bioanalyzer were used for size selection, quantification, and library quality control. Quantified libraries were sequenced on an Illumina platform (NovaSeq 6000) according to the manufacturer’s instructions. Raw sequencing data were aligned to the human reference genome (GRCh38/hg38) using Hisat2 v2.0.5. For quantification, featureCounts v1.5.0-p3 was used to count the read numbers mapped to each gene, and the fragments per kilobase of exon per million mapped fragments (FPKM) of each gene was calculated based on the length of the gene and read count mapped to this gene (Table S2). Differentially expressed genes (DEGs) in the two conditions/groups were analyzed using the edgeR package (3.22.5). The ClusterProfiler R package was used for enrichment analysis (Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway). DEG analysis was performed using an adjusted p-value (Padj) <0.05 as a 2-fold cutoff.

RNA-seq libraries were prepared using the NEBNext Ultra II RNA library preparation kit for Illumina (NEB E7770S) in conjunction with the NEBNext poly(A) mRNA magnetic isolation module (NEB E7490) and NEBNext multiplex oligos for Illumina (NEB E7335 and E7500). For library preparation, we used 10 ng of TRAP RNA from Tg(Atoh1-Egfp-L10a) mice, 800 ng of TRAP RNA from Tg(NeuroD1-Egfp-L10a) mice, or 50–500 ng of total RNA from cultured GCs. Input samples for each developmental age in the TRAP experiment were pooled in equal amounts to yield one input per time point. The quality of the sequencing libraries was evaluated using the Agilent 2200 TapeStation with D1000 High-Sensitivity ScreenTape. The samples were sequenced at the Rockefeller University Genomics Resource Center using a NextSeq 500 sequencer (Illumina) to obtain 75-bp single-end reads.