Nebnext poly a mrna magnetic isolation module kit

Gemmae of WT, Mpaccb8a-1/b-4, and Mpaccb8a-2/b-3 were incubated on half-strength Gamborg’s B5 medium for 2 weeks at 22 °C under continuous light. After 2 week-culture, the thalli were transferred to the medium with or without phosphate. Three biological replicates were prepared for each genotype. Total RNA was extracted using the NucleoSpin RNA Plant kit (Macherey-Nagel, Germany). mRNA was isolated from the total RNA with an NEBNext poly(A) mRNA Magnetic Isolation Module Kit (New England Biolabs, MA, USA). RNA-seq libraries were synthesized using an NEBNext Ultra RNA Library Prep Kit for Illumina and an NEBNext Adaptor for Illumina Kit (New England Biolabs, MA, USA). Paired-end sequencing was performed using the HiSeqXten (Illumina, CA, USA).
The reads were mapped onto the Marchantia paleacea genome^{3 (link)} assembly using HISAT2^{72 (link)} for Galaxy (https://usegalaxy.org) with default parameters. Read counts were calculated by Feature Counts for Galaxy. To identify differentially expressed genes, q values were calculated by the TCC R package^{73 (link)}, and genes with q value <0.05 and log2-fold change >1 or log2-fold change <−1 were selected as up-or down-regulated genes. Summary statistics of RNA-seq analysis are available in Supplementary Data 3.

Total RNAs were isolated from embryos at the indicated stages using TRIzol reagent (ThermoFisher, 15596026). The mRNAs were purified using a NEBNext Poly(A) mRNA Magnetic Isolation Module Kit (NEB, E7490) according to the user manual. Illumina Truseq libraries were constructed according to the manufacturer’s instructions and sequenced using NovaSeq platform.

Trizol reagent (Tiangen, Beijing, China) was used to extract total RNA from undifferentiated and differentiated chicken preadipocytes (Add 1 mL of Trizol per 2.5 × 10⁶ cells). The NEBNext Poly(A) mRNA Magnetic Isolation Module kit (New England Biolabs, Ipswich, MA, USA) and NEBNext Small RNA Library Prep Set for Illumina (Multiplex Compatible) kit (New England Biolabs, Ipswich, MA, USA) were used to construct the lncRNA libraries and miRNA libraries following the manufacturer’s protocol, respectively. Six lncRNA libraries (three for each group) and six miRNA libraries (three for each group) were constructed. The 12 constructed libraries were sequenced using the Illumina HiSeqTM 4000 sequencing platform (Illumina Inc., San Diego, CA, USA).

After surgical resection, tissue was firstly stored in RNAlater RNA stabilization reagent (Catalog No. 76106, QIAGEN, Dusseldorf, Germany) and kept on ice. Total RNA was extracted with the RNeasy Mini Kit (Catalog No. 74104, QIAGEN) following the manufacturer’s instructions. Concentration of RNA was quantified with the NanoDrop instrument (ND-2000, ThermoFisher Scientific, Waltham, MA), and quality of RNA was evaluated with fragment analyzer (AATI, Palo Alto, CA). Libraries were constructed using NEBNext Poly(A) mRNA Magnetic Isolation Module Kit (Catalog No. E7490L, NEB, Ipswich, MA) and NEBNext Ultra RNA Library Prep Kit (Catalog No. E7770, NEB), and sequenced on Hiseq 4000 (Illumina) in paired-end 150 bp.

On the 21st day of osteogenic differentiation, the total RNA from the OM and the 100 μg/mL LF groups were isolated using Trizol Reagent. NEBNext^® Poly(A) mRNA Magnetic Isolation Module kit (NEB, Ipswich, MA, USA) was used to separate the mRNA from 1 μg of RNA, and NEBNext^® UltraTM II mRNA Library Prep Kit (NEB, Ipswich, MA, USA) was used to create the mRNA library. The Novaseq 6000 instrument and Novaseq S4 reagent kit were used for library sequencing. Using the DESeq R program (1.18.1), differential expression analysis of two groups was carried out. DESeq offers statistical techniques for detecting differential expression in digital gene expression data using a model based on the negative binomial distribution. The p-values that resulted from this were modified using Benjamini and Hochberg’s method for reducing the false discovery rate. Genes identified by DESeq with |log2(FoldChange)| > 1 and an adjusted p value < 0.05 were classified as differentially expressed. We implemented KEGG enrichment analysis and visualized the differentially expressed genes using the cluster profile R package. Differentially expressed genes were thought to be highly enriched if the adjusted p-value was <0.05.