title: NextSeq 500 paired end sequencing; RNA-seq of zebrafish sa12692 mutants against WT controls
design: RNA-seq of zebrafish sa12692 mutants against WT controls
library_construction_protocol: Larvae were euthanized by rapid freezing. Ten larvae were pooled per sample and homogenized in TRIzol®. All centrifugation steps for RNA extractions occurred at 4°C. RNA was extracted using the TRIzol® method. Pools of 10 zebrafish larvae (per biological replicate) were collected at 5 dpf and placed in a 1.5 mL tube. Two adult hearts were pooled per biological replicate; these were transferred from RNAlater® to a 1.5 mL tube. TRIzol® (250 μL) was added to each tube. The samples were homogenized thoroughly using a sterile 21G needle and a 1 mL syringe. Samples were incubated for 5 minutes at room temperature (RT). Chloroform was added (one fifth of the volume of TRIzol® used) followed by immediate agitation for 15 seconds and incubation at RT for 3 minutes. For phase separation, samples were centrifuged at 12000 g for 20 min. The upper aqueous phase containing the RNA was removed carefully and transferred to a new tube. Isopropanol was added (half the volume of TRIzol® used) and mixed gently. Samples were incubated for 10 minutes at RT and centrifuged at 18000 g for 10 minutes. The supernatant was removed and the pellet was washed in 70% ice-cold ethanol (one tenth of the volume of TRIzol® used) followed by centrifugation at 18000 g for 10 minutes. The ethanol was removed and the pellet was allowed to dry at RT for ~7 minutes. The pellet was resuspended in 88 µL of nuclease-free water and heated at 55°C for 7 minutes to fully resuspend the pellet. To remove any remaining DNA, 2 µL (4 units) of DNase enzyme and 10 µL of 10X DNase buffer were added and the samples were incubated for 10 minutes at 37°C. Phenol (200 µL) and 20 µL of sodium acetate were added and the samples were centrifuged at 18000 g for 5 minutes. The upper phase was transferred to a new tube and 200 µL of chloroform:isoamyl alcohol (24:1) was added, followed by centrifugation at 18000 g for 5 minutes. The chloroform:isoamyl alcohol step was repeated. The upper phase was transferred to a new tube and 200 µL of isopropanol was added to precipitate the RNA. The samples were incubated at -80°C for 30 minutes followed by centrifugation at 18000 g for 30 minutes. The isopropanol was discarded and 200 µL of ice-cold ethanol was added to wash the pellet. The samples were centrifuged for a final 5 minutes at 18000 g. The ethanol was discarded and the pellets were air-dried for 5-10 minutes. The samples were resuspended in 20 µL nuclease-free water. The RNA was stored at -80°C. The quality/integrity of the RNA was assessed with the use of an Agilent RNA 6000 nano kit on an Agilent Bioanalyzer 2100, by following the manufacturer's instructions. RNA integrity was measured by a software tool on the Agilent Bioanalyzer which calculates an RNA integrity number (RIN) for each sample. Only RINs ≥8 were accepted for library synthesis. RNA concentration and purity was assessed on a Qubit Fluorometer using the Qubit® RNA HS (High Sensitivity) Assay Kit, according to the manufacturer's instructions. The values obtained for RNA concentration were used to calculate the amount needed for the library preparation. Total RNA was converted to a cDNA library using the Illumina TruSeq® Stranded mRNA Library Preparation Kit, following the manufacturer's instructions. An RNA input of 1000 ng was used for larval samples and 600 ng for adult heart samples. The only minor but important deviation from the protocol was a shorter drying time for the magnetic beads due to the temperature of the lab environment. The recommended drying time of 15 minutes was reduced to 10 minutes. Briefly, the polyA-containing mRNA molecules were purified and fragmented into smaller pieces. First strand cDNA was synthesised from the cleaved RNA fragments using reverse transcriptase and random primers. The RNA fragments were removed and double-stranded cDNA was synthesised with the incorporation of dUTP instead of dTTP in the second strand. Strand specificity was achieved by degradation of the second strand as the polymerase used does not recognise past the dUTP nucleotide. The 3' ends of the cDNA fragments were adenylated to prevent them ligating to each other. Complementary indexing adapters were ligated to the ends of the fragments to allow hybridisation of the fragments to the flow cell at the sequencing stage. A final PCR step ensured enrichment of cDNA fragments with adapters at both ends and amplification of the library. The size and purity of each library sample was assessed on an Agilent Bioanalyzer using the Agilent DNA 1000 Kit. The DNA of each library sample was quantified on a Qubit Fluorometer using the Qubit dsDNA HS (High Sensitivity) Assay Kit, according to the manufacturer's instructions. All 12 samples were pooled at equal concentration and the quality of the final product was validated on an Agilent Bioanalyzer. The average fragment size was 270 bp.