Nextflex rapid directional rna seq kit

Metabolic labeling of cells for purification of nascent transcript was carried out as described (Rädle et al., 2013 ). Cells were treated with tamoxifen for the times indicated and pulse labeled with 500 μM 4-thiouridine for 15 minutes prior to harvesting in Trizol (Life Technologies) for RNA purification. For isolation of total RNA for sequencing, cells were harvested in Trizol and processed according to manufacturer’s instructions.
Libraries for sequencing were prepared using the NEXTflex Rapid Directional RNA-seq kit (Illumina) or SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian (Takara Bio) and sequenced as above. Individual expression assays were carried out using gene-specific TaqMan probes (Life Technologies).
RNA-seq libraries were mapped against the mouse reference genome mm10 using GSNAP (gmap-2014- 12-17) (Wu and Nacu, 2010 (link)) with parameters “–m 7 –i 1 –N 1 –w 100000 –E 100 –n 10.” Gene read counts were calculated with HTSeq (v0.6.1) based on gene annotation from Ensembl release 75, and normalization and differential expression analysis were performed using the R package DESeq2 (Love et al., 2014 (link)) with the default model. We identified differentially expressed genes at FDR-adjusted p-values less than 0.05. For time series analyses differential expression was assessed for all pairwise comparisons against the 0 h time point.

Libraries for sequencing were prepared using the NEXTflex Rapid Directional RNA‐seq kit (Illumina) or SMARTer^® Stranded Total RNA‐Seq Kit v2—Pico Input Mammalian (Takara Bio) and sequenced on the Illumina platform as for ChIP‐seq libraries. Low‐quality reads and adaptor sequences were removed by Trim Galore! (version: 0.4.1), and the trimmed reads were aligned to mouse reference genome (GRCm38/mm10) using Tophat (version v2.1.0; Kim et al, 2013). Read counts per gene were obtained using featureCounts (version 1.5.0; Liao et al, 2014) with annotations from Ensembl release 86 (Yates et al, 2016). Normalisation, with and without spike‐ins, and differential expression analyses were performed using the R/Bioconductor Deseq2 package (version 1.14.1; Love et al, 2014). The R prcomp function was used for principal component analysis (PCA). Gene Ontology (GO) analysis was performed using DAVID (da Huang et al, 2009b).