Truseq rna protocol

For the RNA-seq experiment, the K. xylinus E25 strain was cultured in 10-mL test tubes containing 5 mL of a culture medium at 30 °C under static conditions for 4 days. In total, 6 cultures were prepared, 3 with SH medium and 3 with SH medium containing 1% ethanol. To the culture was added a 1% of cellulase solution (v/v) (from Trichoderma reesei ATCC 26921, Sigma-Aldrich, Steinheim, Germany). The samples were then incubated for 3 h at 30 °C under static conditions with occasional vortexing. The released cells were harvested. Total RNA was purified using a RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. The sequencing libraries were prepared according to the TruSeq RNA protocol (Illumina) by BioNanoPark Łódź, Poland. Sequencing was performed in the pair-end mode (75 cycles) using a NextSeq500 sequencer (Illumina). Between 17 and 20 million reads were generated per library. The raw reads were deposited at GenBank under BioProject ID: PRJNA498189.

All experiments with Tetraodon were performed in accordance with guidelines and regulations covered and approved by the Home Office Project Licence 44518. Breeding of fishes, extraction of eggs and collection of embryos was performed as described in a recent report20 (link). Total RNA was extracted with Trizol (Invitrogen) according to the manufacturer’s protocol from eggs, whole embryo at 30% epiboly (30 epi) and whole embryo at 24 hours post fertilisation (24 hpf). The RNA samples were treated with 2U Dnase I (Qiagen) per μg RNA sample at 37 °C for 10 minutes. Digested samples were then treated with 20 mg/mL proteinase K (Sigma Aldrich) at 37 °C for 45 minutes. The quality and quantity of total RNA were assessed with the Bioanalyzer 2100 (Agilent) and no sign of degradation was detected (RIN > 9.0). Sequencing libraries were generated from total RNA samples following the Truseq RNA protocol (Illumina). Single end reads (1 × 50 nucleotides) were obtained from 3 lanes on a Hiseq1000 using SBS v3 kits (Illumina). Cluster detection and base calling were performed using RTAv1.13 (Illumina). Quality of reads was assessed with CASAVA v1.9. Sequencing reads with a mean Phred score higher than 37 were further considered for mapping and assembly.

C-33 cells were seeded at a density of 1×10⁶ cells per 10 cm dishes and grown for 2 days. E2 expression was induced by the addition of 3 µM CdSO₄ for 4 h. Total RNA was purified using RNeasy (Qiagen), and analyzed for integrity using the Agilent RNA 6000 nano kit on 2100 Bioanalyzer (Agilent). Both polyadenylated and non-polyadenylated (after rDNA subtraction) RNA was sequenced. Two different libraries were constructed for each sample. For one library, total RNA was purified by poly A selection following manufacturer's instructions. For the second library, 1.5 µg total RNA was rRNA depleted using Ribo-Zero (Epicentre, Madison, WI), followed by library generation using the Illumina TruSeq RNA protocol, beginning at the fragmentation step. Libraries were sequenced on an Illumina GAIIx. The adapters were trimmed from raw sequences and low quality reads were filtered out. Processed reads were mapped to human genome assembly hg19 using Tophat and differentially expressed gene analysis was performed using Cufflinks [77] (link). Data was visualized using the Integrative Genomics Viewer (Broad Institute). The dataset can be accessed at GEO: GSE52367.

RNA extractions were carried out using RNeasy Spin columns (Qiagen) following the manufacturer’s instructions for animal tissues using the optional on-column DNase I digestion (Qiagen). RNA quality was assessed using a Nanodrop spectrophotometer (NanoDrop products) and a Bioanalyser (Agilent Technologies). RNA samples with RNA integrity number values from 8 to 10, absorbance ratios of 260:280 nm greater than 2 and 260:230 nm between 2 and 2.2 were used for sequencing using the service provided by The Genome Analysis Centre (TGAC, Norwich, UK). In brief, libraries were constructed using the PerkinElmer Sciclone with the TruSeq RNA protocol (Illumina). mRNA was separated from 1 μg of total RNA by poly-A pull down using biotin beads. mRNA was fragmented, cDNA synthesized, and overhanging ends repaired to create blunt ended DNA. 3′-A overhangs were added using Taq DNA polymerase, and ligated to corresponding 3′-T overhangs present on adapter sequences. Unligated adapters were removed using size selection XP beads (Beckman Coulter). The nine libraries, each prepared with different barcoded adapters, were normalized and pooled, spiked with 1% PhiX control v3, and sequenced on a single lane of Illumina HiSeq2000 using 100 cycles for each paired-end read.