For the pull‐down assay, a TranscriptAid T7 Kit (Thermo Fisher, K0441) and a RNA‐Protein Pull‐Down Kit (Thermo Fisher, 20164) were used in accordance with the instructions. Briefly, HHAS1 and antisense transcripts were transcribed and biotin‐labeled. Then, the labeled RNAs were incubated with BMSC lysates, and the pulled‐down proteins were extracted for further detection.
Transcriptaid t7 kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The TranscriptAid T7 Kit is a tool used for in vitro transcription. It facilitates the synthesis of large amounts of RNA from DNA templates containing a T7 promoter sequence.
Automatically generated - may contain errors
Lab products found in correlation
Rna protein pull down kit, by Thermo Fisher Scientific (1 mentions)
Pierce streptavidin magnetic beads, by Thermo Fisher Scientific (1 mentions)
Phenol red, by Merck Group (1 mentions)
Picoliter microinjector, by Warner Instruments (1 mentions)
Alexa fluor 488 protein labeling kit, by Thermo Fisher Scientific (1 mentions)
Phusion flash polymerase, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 555, by Thermo Fisher Scientific (1 mentions)
Cas9 protein, by PNA Bio (1 mentions)
5 protocols using transcriptaid t7 kit
1
Biotin-labeled RNA Pull-Down Assay
2
Biotinylated RNA Pull-Down Assay
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The biotinylated RNA pull-down experiment was implemented according to previous studies [57 (link)]. Biotinylated circMYOF probes were synthesized and labelled by a TranscriptAid T7 Kit (Thermo Fisher Scientific, USA) in accordance with the manufacturer’s instructions. Then, probe-coated beads were produced using PierceTM streptavidin magnetic beads (Thermo Fisher Scientific, USA). Cells were collected and then hatched with streptavidin magnetic beads at 4 °C for 4 h. Afterwards, the binding RNA was isolated with RNAiso Plus. RNA bands were analysed by agarose gel electrophoresis.
3
Zebrafish GARS Knockdown Protocol
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Morpholinos targeting the 5ʹ untranslated region of zebrafish GARS, the exon 8 donor splice site, and scrambled controls were selected and synthesised by Gene Tools (http://www.gene-tools.com ). GARS 5′ untranslated: 5′-TGCGCTACACAGAGACAGCATGGAC-3′, GARS 5′ untranslated scrambled control: 5′-TGCcCTAgACAcAGACAcCATcGAC-3′, GARS exon8: 5′-AGTCAGTTGTAATCCACACCTAACA-3′, GARS exon 8 scrambled control: 5′-AGTgAcTTcTAATCgAgACCTAACA-3′. Approximately 1 nl of 6 pg/nl morpholino in 0.05% phenol red (Sigma, St Louis, MO, USA) was injected into embryo yolks at the one to four cell stage.
Full-length D. rerio GARS cDNA was obtained from Thermo Bioscience (Pittsburgh, PA, USA), amplified by PCR with BamH1 and Xho1 ends, and inserted into a pCDNA6 vector 3′ of a 6myc tag (6 concatenated copies of the c-myc epitope). The pCDNA6-GARS vector was linearised and capped RNA synthesised using the Thermo Scientific TranscriptAid T7 kit (#K0441). Poly(A) tailing was done with the Ambion Poly(A) tail kit (AM1350, Ambion, Waltham, MA, USA). Approximately 1 nl of 250 ng/μl RNA was injected into the embryo yolk.
Glass microinjection needles were made by pulling 1-mm capillary filament (World Precision Instruments, Sarasota, FL, USA) with a Pul-1 Micropipette Puller (World Precision Instruments). Injections were made with a Picoliter Microinjector (Warner Instruments PLI-100A) set to 10 to 20 psi.
Full-length D. rerio GARS cDNA was obtained from Thermo Bioscience (Pittsburgh, PA, USA), amplified by PCR with BamH1 and Xho1 ends, and inserted into a pCDNA6 vector 3′ of a 6myc tag (6 concatenated copies of the c-myc epitope). The pCDNA6-GARS vector was linearised and capped RNA synthesised using the Thermo Scientific TranscriptAid T7 kit (#K0441). Poly(A) tailing was done with the Ambion Poly(A) tail kit (AM1350, Ambion, Waltham, MA, USA). Approximately 1 nl of 250 ng/μl RNA was injected into the embryo yolk.
Glass microinjection needles were made by pulling 1-mm capillary filament (World Precision Instruments, Sarasota, FL, USA) with a Pul-1 Micropipette Puller (World Precision Instruments). Injections were made with a Picoliter Microinjector (Warner Instruments PLI-100A) set to 10 to 20 psi.
4
Cas9 Protein Production and Validation
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All Cas9 proteins used in this study were derived from the prototypic Streptococcus pyogenes species. Cas9 protein was produced as described in Seamon et al.33 (link) and contained a N-terminal nuclear localization signal and a C-terminal HIS tag. For in vivo studies, endotoxin-free Cas9 was purchased from IDT (IDT, 1081058). For microscopy experiments using labelled Cas9, the AlexaFluor 488 protein-labeling kit was used per manufacturer protocols (ThermoFisher, A10235). Re-purified Cas9-488 was assayed to confirm enzymatic activity in vitro. In vitro transcription of gRNA was performed according to https://www.protocols.io/view/In-vitro-transcription-of-guide-RNAs-dwr7d5 , using Phusion Flash Polymerase (ThermoScientific), TranscriptAid T7 Kit, and MEGAClear Transcription Clean-Up Kits (Thermo Scientific). For the Stoplight/AAVS1 targeting-gRNA, the guide sequence was 5′-GGGGCCACTAGGGACAGGAT-3′. For targeting the Ai9-Reporter locus, end-modified guide was purchased from Synthego using the guide sequence of 5′-AAGTAAAACCTCTACAAATG-3′ reported in Staahl et al.37 (link). The NPC1 guide-target was chosen by empirically screening 4 Cas9 candidate guides with targets in Exons 6 and 8 for efficacy, with the best guide being pursued. The selected guide (hNPC1-Exon8-gRNA1) for LCMSN studies was purchased from Synthego, with the sequence 5′-AGTATCTGTATGTCAAGCGG-3′.
5
Generating X. tropicalis Transgenic Embryos
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Wild-type X. tropicalis frogs were purchased from NASCO, and the snai2:eGFP transgenic line was generated previously23 (link). mRNAs encoding desired proteins were generated by in vitro transcription. For embryo production, female and male frogs were each primed with 20 IU human chorionic gonadotropin (HCG), and boosted with 200 IU HCG the next day for natural mating. Fertilized embryos were collected and injected in one blastomere at 2-cell stage; the uninjected blastomere served as a control. Alexa Fluor 555 (Invitrogen) or ß-galactosidase mRNA was co-injected as a lineage tracer. Morpholino for DPH1 KD (5’-CTTCCGCCATCTCTGACATATTTA-3’) was designed and synthesized by Gene Tools. The guide RNAs for CRISPR/Cas9 mediated DPH1 KO in X. tropicalis were designed using the CRISPRscan software52 (link). Three individual sites were designed for CRISPR targeting. Protospacers g1 (5′-GTGATGGGCGATGTGACGTA-3′), g2 (5′- GGTTGAAAGTTGAAGCGAA-3′) and g3 (5′- GGCATCAATCGGGACTGAG-3′) were embedded in forward primers. The DNA templates for gRNA transcription were obtained by PCR using pUC57-T7-gRNA scaffold vector as a template (see Supplementary Table 3 for primer sequences), and gRNAs were generated by in vitro transcription using the TranscriptAid T7 kit (Thermo Scientific K0441). To knock out dph1, each X. tropicalis embryo was injected with 1 ng Cas9 protein (PNA Bio) and 300 pg gRNA.
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