Next ultra rna library prep kit

To explore the regulatory mechanism of α-CBD on HepG2 cells at the molecular level, HepG2 cells were treated with 20 mg/L α-CBD for 48 h (experimental group). The total RNA of cells from control and experimental groups was extracted using the Cell RNA Kit (NEB, Ipswich, MA, USA). Subsequently, 5 μg of RNA from each sample was used to construct libraries using the NEB Next Ultra RNA Library Prep Kit from Illumina, according to the manufacturer’s instructions. Differential gene expression analysis between the experimental and the control groups was performed using the DESeq R package (1.18.0). We mainly focused on the function of differentially expressed genes. RNA-Seq data is deposited under the accession codes PRJNA516386, PRJNA516390, PRJNA516391, PRJNA516392, PRJNA516393 and PRJNA516394 in the NCBI SRA database.

RNA sample was used for library preparation using NEB Next Ultra RNA Library Prep Kit for Illumina Indices were included to multiplex multiple samples. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first strand cDNA was synthesized using random hexamer primers followed by the second strand cDNA synthesis. The library was ready after end repair, A-tailing, adapter ligation, and size selection. After amplification and purification, insert size of the library was validated on an Agilent 2100 and quantified using quantitative PCR (Q-PCR). Libraries were then sequenced on Illumina NovaSeq 6000 S4 flowcell with PE150 according to results from library quality control and expected data volume. Library preparation/sequencing/analysis is performed by Novogene (UK) Company Limited.

RNA sample with a RIN value above seven was used for cDNA synthesis. Equal amounts of high-quality RNA from accessory gland tissues of virgin males and males interrupted during mating were taken for cDNA synthesis. The cDNA library construction was done using NEB Next Ultra RNA Library Prep Kit and Illumina sequencing of the samples was performed at Sandor Life Sciences Pvt. Ltd. (Hyderabad, India). The mRNA was purified from 1 μg of the total RNA using Oligo dT beads and was fragmented into short sequences at the appropriate temperature. These fragments served as templates and the first strand of cDNA was synthesized using random primers and reverse transcriptase, followed by the synthesis of the second-strand cDNA using RNaseH and DNA polymerase I. After end-repair, the fragments were ligated using sequencing adaptors and purified using AMPure XP beads. These adaptor-ligated fragments were amplified using PCR and the products were further purified using AMPure XP beads to create a cDNA library and were assessed on the Agilent Bioanalyzer 2100 system. Quantification and size distribution of the prepared library was determined using a Qubit flourometer and Agilent Tape station D1000 Kit according to the manufacturer’s instructions. The resulting cDNA libraries were then paired-end sequenced (2 × 150 bp) with the Illumina HiSeq^TM platform.

Total RNA was harvested from day 3 NMPs obtained from TBXT shRNA sOPTiKD hESC in the presence and absence of Tet (three biological replicates) using the total RNA purification plus kit (Norgen BioTek) according to the manufacturer’s instructions. Sample quality control, library preparation, and sequencing were carried out by Novogene (http://en.novogene.com). Library construction was carried out using the NEB Next Ultra RNA Library Prep Kit and sequencing was performed using the Illumina NoveSeq platform (PE150). Raw reads were processed through FastQC v0.11.2 and Trim Galore. Reads were aligned using STAR v2.4.2a (Dobin et al., 2013 (link)) to the human reference genome assembly GRCh38 (Ensembl Build 79) in the two-pass mode. The RSEM v1.3.0 (Li and Dewey, 2011 (link)) was used to extract expected gene counts, where genes expressed in <3 samples or with total counts ≤5 among all samples were excluded. We identified genes showing significant differential expression with DESeq2 (Love et al., 2014 (link)), with log2FoldChange>|0.5| and Benjamini–Hochberg-adjusted p<0.05. Data were deposited to GEO (Accession number: GSE184620).

Non-directional RNA-seq was performed at Novogene (Cambridge, UK) using the NEB Next Ultra RNA Library Prep Kit for mRNA-seq library preparation followed by sequencing with the Illumina Novaseq 6000 platform with a PE150 sequencing strategy.

E13.5 embryos were dissected from Tie2Cre;Ino80 fl/fl pregnant females. RNA from whole hearts were used to prepare sequencing libraries with NEB Next Ultra RNA library prep kit and sequenced with 76 bp Illumina paired-end reads. Raw sequencing library quality was assessed using FastQC. Reads were then mapped to genome GRCm38 (mm10) using STAR (v2.4.2a) with gene annotations from the GENCODE primary assembly (vM6). Reads aligning to transcripts were counted using the summarizeOverlaps function from the GenomicAlignments R package^{54 (link)}. An average of 33.9 M reads were counted for each WT replicate, vs. an average of 53.5 M reads for each KO replicate. Count data were input into DESeq2, and data were regularized-log-transformed prior to visualization by heatmap^{55 (link)}. Significance results and log₂(fold change) values were generated using the DESeq2 algorithm on untransformed count data.
Gene ranks for pre-ranked GSEA were defined as -log₁₀(padj) for upregulated genes in the mutant (log₂(fold change)>0) and (−1) × −log₁₀(padj) for downregulated genes, where padj is the Benjamini-Hochberg corrected p-value from DESeq2. This is analogous to the ranking system used in reference^{56 (link)}. Enrichment of 15597 genes with non-zero expression was evaluated within 7057 hallmark gene sets^{57 (link)} and Zhang et al.^{37 (link)} using the “weighted” enrichment statistic.