GSM7157754: flk:GFP,injected with MV4-11 derived Evs 3; Danio rerio; RNA-Seq
Five hundred of Tg(flk:GFP) larvae were injected with PBS (control) or EVs and three replicates were prepared for each treatment. The larvae were then anesthetized at 24 h-post-injection (hpi) before cutting tails on ice. The tails were collected and gently triturated in 1.5 mL cold trypsin solution (0.5 g/L; T3924; Sigma) with 1,000 μL pipette tip for 20 min. The dissociated cells were then filtered with a 70 μm filter (BD Transduction Laboratories) to remove the undissociated tissue and washed twice with cold PBS. Cells were then resuspended in 2.5 mL PBS and stained with 7-AAD (5 μg/mL, BD Transduction Laboratories) on ice for 30 min. About 1X10^5 GFP+ cells were sorted by FACS (MoFlo XDP, Beckman) and total RNA was extracted with Trizol (15596, Invitrogen) according to the manufacturer's instructions. Low input RNA-Seq was performed according to the published protocol with modifications . Briefly, mRNAs were purified by VAHTS RNA Clean Beads (N412, Vayzme) in RNase-free H2O with reverse transcription mix, amplified for 15 cycles, and reverse transcribed for cDNAs before purifying with VAHTS DNA Clean Beads (N411, Vazyme). Enriched cDNA was quantified using Qubit dsDNA HS Assay Kit (Q3285, Guangzhou IGE biotechnology ltd) and verified by DNA gel electrophoresis (1.5-2 kb). Libraries were prepared by using TruePrep DNA library Prep Kit V2 for Illumina (TD503, Vazyme) and adaptor-ligated DNAs (400 bp) were selected by VAHTS DNA Clean Beads (N411, Vazyme). Libraries were sequenced by using NovaSeq-PE150 as 150 bp paired-end.