Truseq stranded mrna

Triplicate RNA-Seq samples were collected for each developmental stage, one from each independent tank. For each sample, exactly 200-staged-matched embryos were snap frozen in a 70% ethanol dry ice slurry and stored at –80°C. Total RNA was extracted from each sample as per manufacturer's recommendations (Relia Miniprep kit, Promega) and tested for quality and quantity using Nanodrop and Agilent Tapestation. All samples had an RNA integrity number of over 7. Libraries were prepared by the DNA sequencing facility in the Biochemistry Department at the University of Cambridge (TruSeq Stranded mRNA, Illumina) and sequenced on an Illumina NextSeq500 generating over 300 million 150 bp stranded paired-end reads. All raw data are publicly available from NCBI SRA accession number: PRJNA803976.

The total RNA from the tissue was isolated by TRIzol reagent. Samples were analyzed in the Roswell Park Comprehensive Cancer Center Genomics Shared Resource. TruSeq Stranded mRNA (Illumina, San Diego, CA, USA) was used for library preparation. Raw sequencing reads were aligned to the human genome (GRCh38.74) and mouse genome (Mm10) using STAR [63 (link)] (40.4×10⁶ –69.0×10⁶ uniquely mapped reads per sample). Raw feature counts were normalized, and differential expression analysis was conducted using DESeq2 [60 (link)]. Differential expression rank order was utilized for subsequent Gene Set Enrichment Analysis (GSEA), performed using the clusterProfiler package in R. Gene sets queried included the Hallmark, canonical pathways, and GO Biological Processes Ontology collections available through the Molecular Signatures Database (MSigDB) [64 (link)]. Further, differentially expressed genes underwent transcriptional metabolic assessment [36 (link)], to determine which metabolic pathways are most highly transcriptionally dysregulated to a statistically significant extent.

Thyroid glands from 4-week-old WT (n = 4) and Glis3-Pax8Cre (n = 4) mice fed an ND or a LID for 6 days were collected and RNAs isolated with a RNAqueous-Micro total RNA isolation kit (ThermoFisher). mRNA isolation and library generation were carried out with a TruSeq Stranded mRNA and TruSeq RNA Library preparation kits (Illumina Inc., San Diego, CA), respectively. Sequence was read by paired-end sequencing using a NextSeq500 or NovaSeq 6000 Sequencing System (Illumina). Raw reads pairs were filtered to retain only those with average base quality score > 20 for both read ends. Filtered read pairs were mapped to the mm10 reference assembly via STAR v2.5 [41 (link)] with parameters “--outSAMattrIHstart 0 --outFilterType BySJout --alignSJoverhangMin 8 --limitBAMsortRAM 55000000000 --outSAMstrandField intronMotif --outFilterIntronMotifs RemoveNoncanonical”. Counts per gene were extracted via featureCounts (Subread v1.5.0-p1) [42 (link)] with parameters “-s0 -Sfr -p” for RefSeq gene models as downloaded from the UCSC Table Browser on November 7 2017. Differential gene expression analysis was performed using DESeq2 v1.14.1 [43 (link)]. For the purposes of pathway analysis, differentially expressed genes are defined at FDR threshold 0.01 and fold change > 2 or < −2.

After sorting, the low-MYC and high-MYC cells were seeded overnight. Considering the effects of MYC on global gene expression, external RNA spike-in control (ERCC, Thermo Fisher Scientific) was added to cells in proportion to cell count before total RNA extraction. The mRNA libraries were prepared using TruSeq Stranded mRNA (Illumina) and sequenced on a NextSeq sequencer.

Liver and parietal lobe of the cerebrum tissues from two healthy Thoroughbred mares was obtained from the functional annotation of the animal genome biobank [20 (link)] to investigate lncRNA expression in both a homogeneous and a complex tissue. RNA was isolated using a phenol-chloroform method with a column clean up. RNA quality was measured using an Agilent Bioanalyzer (RIN = 8.7). Two RNA-seq libraries were prepared from each tissue sample, one based on poly-A⁺ selection and the other using rRNA-depletion. Poly-A⁺ selected libraries were made using a strand-specific poly-A⁺ capture protocol (TruSeq Stranded mRNA, Illumina, San Diego, CA, USA). A bioanalyzer was used to ensure all poly-A⁺ selected libraries had adequate size distributions. The rRNA was depleted (Ribo-Zero, Illumina, San Diego, CA, USA) and prepared as strand-specific (TruSeq Stranded Total RNA Library pre kit, Illumina, San Diego, CA, USA). The libraries were size selected for 140 bp ± 10% fragments and sequenced on a HiSeq 4000 to an average depth of 30 M mapped reads (ERX2600970, ERX2600971). The reads are paired end and 125 bp long.