Pre-mRNA templates were transcribed using the MEGAscript T3 Transcription Kit according to the manufacturer’s instructions (Thermo Fisher No. AM1338). In vitro splicing reactions were assembled in 25 μl volumes, each containing 10 μl of splicing extract, 5 μl of 5x splicing buffer (12.5 mM MgCl2, 15% PEG 8000, 300 mM KH2PO4 at pH 7.2, and 30 μl nuclease-free water), 5 μl pre-mRNA template from in vitro transcription, 0.5 μl 200 mM ATP, 1 μl RNaseOUT Recombinant RNase Inhibitor (Life Technologies No. 10777–019), and 3.5 μl nuclease-free water. For reactions involving the addition of regulatory element RNA, this RNA was added to reactions in lieu of water to the final concentrations indicated in the figures. Reactions were incubated at 25°C for 40 minutes and then stopped by the addition of 75 μl nuclease-free water and 100 μl of RNA buffer followed immediately by phenol-chloroform RNA extraction. The extracted RNA was DNase-treated and then used for RT-PCR analysis (see below). Equal amounts of DNase-treated RNA were used for all samples for the cDNA synthesis reactions.
Megascript t3 transcription kit
Manufactured by Thermo Fisher Scientific
The MEGAscript T3 Transcription Kit is a laboratory product designed for in vitro transcription of RNA. It contains the necessary components to synthesize high yields of RNA from DNA templates using the T3 RNA polymerase.
Automatically generated - may contain errors
Lab products found in correlation
Rnaseout recombinant rnase inhibitor, by Thermo Fisher Scientific (1 mentions)
T4 rna ligase 2, by New England Biolabs (1 mentions)
Megaclear transcription clean up kit, by Thermo Fisher Scientific (1 mentions)
Nanodrop spectrophotometer, by Avantor (1 mentions)
Nucaway spin column, by Thermo Fisher Scientific (1 mentions)
Rna clean concentrator 5 kit, by Zymo Research (1 mentions)
6 protocols using megascript t3 transcription kit
1
In Vitro Splicing Assay Protocol
2
In vitro Transcription and Radiolabeling of Target RNAs
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In Figs 3D , E, 4F , and EV3D , the template DNA for the in vitro transcription of the Ago3 target RNA was amplified by PCR using primers for the T3 and T7 promoter. The radiolabeled target RNA was transcribed with the MEGAscript T3 Transcription Kit (Thermo Fisher Scientific) and labeled with α‐32P‐UTP. After transcription, the RNA sample was purified by denaturing PAGE. In Fig 3F , Siwi target RNA was radiolabeled using T4 polynucleotide kinase. In Fig 4G , to produce the target RNA, Ago3 target RNA 5′ and radiolabeled Ago3 target RNA 3′ were ligated using T4 RNA Ligase 2 (New England Biolabs) and purified by denaturing PAGE. The sequences of the template DNA used for in vitro transcription and RNAs were described in Table EV1 .
3
HLA-F-AS1 RNA Pulldown Assay
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A vector expressing HLA-F-AS1 and negative control (NC) RNA with T3 RNA transcriptase was used to prepare RNA transcripts through in vitro transcriptions using the Invitrogen MEGAscript T3 Transcription Kit. Purification of in vitro transcripts was performed using the Invitrogen MEGAclear Transcription Clean-Up Kit. After that, 3ʹ end labeling with biotin was performed using the BIO 3ʹ-End Oligonucleotide Labeling Kit (Interchim). The labeled RNA transcripts were further purified using the MEGAclear Transcription Clean-Up Kit, followed by transfection into cells through the methods mentioned above. Cells were harvested 48 h later to perform cell lysis, followed by using Streptavidin agarose beads to isolate RNA complex. Beads were washed to isolate RNA, and RNA samples were subjected to RT-qPCRs to determine the expression levels of MEG3.
4
Whole-Mount In Situ Hybridization of Marine Invertebrate Embryos
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DNA templates for RNA probe synthesis were amplified with reverse primers that contained T3 promoter. Invitrogen MEGAscript T3 Transcription Kit was then used to amplify digoxigenin-labeled RNA from the DNA templates. WMISH was performed as previously described (Ettensohn et al., 2007 (link)), with minor modifications. Embryos were collected fixed at the desired stage and fixed 4% (paraformaldehyde PFA) in ASW for 1 hr at room temperature. The embryos were then washed twice in ASW and permeabilized and stored in with 100% methanol. Embryos were then rehydrated and incubated with 1 ng/µl RNA probe overnight at 55°C. The following day, the embryos were incubated in blocking buffer (1% BSA (bovine serum albumin) and 2% horse serum in PBST (phosphate-buffered saline containing 0.05% Tween-20)) and then in blocking buffer with 1:2000 α-DIG-AP antibody. Excess antibody was washed away and color reaction for alkaline phosphatase was carried out.
5
Efficient RNAi Knockdown of spo-11
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spo-11RNAi was performed by soaking worms with dsRNA (corresponding to nucleotides 593–1,500 of the T05E11 cosmid). Templates were generated by PCR using the following primer pairs containing the T3 promoter (indicated by lower case letters): (1) forward: 5′-aattaaccctcactaaaggGTCGGGCTAATTTAAACATT-3′, reverse: 5′-TGAATCTTCGTGGTACTCGT-3′; and (2) forward: 5′-aattaaccctcactaaaggTGAATCTTCGTGGTACTCGT-3′; reverse: 5′-GTCGGGCTAATTTAAACATT-3′.
Purified DNA (0.1–0.2 µg) was used for in vitro transcription using a MEGAscript T3 Transcription Kit (Ambion) according to the manufacturer's instructions. Total RNA was isolated using TRIzol (TriFast Peqlab; VWR) according to the manufacturer’s protocol and quantified using a Nanodrop spectrophotometer. To anneal the dsRNA, equal amounts (3,000 ng/µl) of the two ssRNAs were incubated in 3× soaking buffer (32.7 mM Na2HPO4, 16.5 mM KH2PO4, 6.3 mM NaCl, and 14.1 mM NH4Cl) at 68°C for 10 min and then at 37°C for 30 min. L4-stage worms were then soaked in dsRNA at a concentration of 1 µg/µl for 24 h and transferred onto NGM plates; apoptotic nuclei were scored 48 h later. Efficient spo-11RNAi was indicated by the presence of 12 univalents in diakinesis nuclei as indicated inFig. S4 E .
Purified DNA (0.1–0.2 µg) was used for in vitro transcription using a MEGAscript T3 Transcription Kit (Ambion) according to the manufacturer's instructions. Total RNA was isolated using TRIzol (TriFast Peqlab; VWR) according to the manufacturer’s protocol and quantified using a Nanodrop spectrophotometer. To anneal the dsRNA, equal amounts (3,000 ng/µl) of the two ssRNAs were incubated in 3× soaking buffer (32.7 mM Na2HPO4, 16.5 mM KH2PO4, 6.3 mM NaCl, and 14.1 mM NH4Cl) at 68°C for 10 min and then at 37°C for 30 min. L4-stage worms were then soaked in dsRNA at a concentration of 1 µg/µl for 24 h and transferred onto NGM plates; apoptotic nuclei were scored 48 h later. Efficient spo-11RNAi was indicated by the presence of 12 univalents in diakinesis nuclei as indicated in
6
In Vitro Transcription and capping
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In vitro transcription with the MEGAscript T3 Transcription Kit (Ambion 1138) was used to produce a 32-nt 5′-triphosphate RNA (pppRNA) with the same sequence as that used in (23 ). pppRNA was purified using a NucAway Spin Column (Thermo Fisher AM10070) and stored at −20°C. To generate radiolabeled GpppRNA, a 40-μl in vitro capping reaction was prepared, containing 10 mM Tris–HCl pH 7.5, 3 mM MgCl2, 1 mM DTT, 0.1 mM EDTA, 20% (v/v) glycerol, 24 pmol pppRNA, 24 pmol [α-32P]GTP (3000 Ci/mmol, PerkinElmer BLU006H250UC) and 2 pmol recombinant His-CE. This reaction was incubated for 3 h at 30°C and then heat-inactivated for 10 min at 65°C. RNA was purified using the RNA Clean & Concentrator-5 kit (Zymo Research R1016), eluting with 30 μl RNase-free water. Yield of purified radiolabeled GpppRNA was measured by scintillation counting relative to input.
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