title: HiSeq X Ten paired end sequencing; RNA-Seq of wilt type and somatostatin 3 CrispR/Cas9 zebrafish mutants treated with dextran sodium sulfate (DSS)
design: RNA-Seq of wilt type and somatostatin 3 CrispR/Cas9 zebrafish mutants treated with dextran sodium sulfate (DSS)
library_name: Sample 3_p
library_construction_protocol: Each sample consisted of a pool of 10 individuals each of 6 days post fertilization wild type (WT) and homozygous mutant (M) sst3-/-, untreated or treated with dextran sodium sulfate (DSS) which were  were frozen at -80C and stored in liquid nitrogen for RNA extraction and sequencing. Zebrafish embryos were obtained from natural spawning and raised until 1 dpf in an incubator (Panasonic, MIR-254-PC) at 28.5°C in recirculating water (60 μg/mL instant ocean sea salts) and supplemented with methylene blue up to 1 dpf. Larvae were grown at 28.5℃ for 5 days and from 6-15 dpf were fed with Paramecium.  RNA from larvae was isolated using TRIzol (Invitrogen), according to the manufacturer's instructions. The purity of the RNA was assessed using the NanoPhotometer spectrophotometer (IMPLEN, CA,USA) and the RNA concentration was measured using the Qubit RNA Assay Kit in Qubit 2.0 Flurometer (Life Technologies, CA, USA). RNA integrity was further assessed using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, CA, USA).   Done by Novogene: A total amount of 3 μg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext Ultra RNA Library Prep Kit for Illumina (NEB, USA) following manufacturer's recommendations and index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer (5X). First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNase H-). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3' ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 250~300 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then 3 μl USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37°C for 15 min followed by 5 min at 95 °C before PCR. ThenPCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system.
attributes.tag: Experimental Factor: genotype
attributes.value: CRISPR/Cas9 mediated Sst3-/- knockout
attributes.tag: Experimental Factor: stimulus
attributes.value: none