Ngn3-Cre-RFP organoids were treated with ISX-9 for 48 h, collected and processed for fluorescence-activated cell sorting as just described. DAPI was added immediately before sorting. RFP-positive, DAPI-negative cells were sorted into 384-well plates containing 384 unique molecular identifier (UMI) barcode primer-sets using a FACS Aria™ II (Beckton Dickinson). Samples in plates were centrifuged and stored at −80 °C. The samples were then processed by Single Cell Discoveries B.V. according the SORT-seq method [33 ]. Briefly, first and second strand synthesis (Invitrogen) was performed and all wells of a single plate were pooled. After in vitro transcription (Ambion), the amplified RNA was reverse transcribed and amplified for 10 to 12 cycles with Illumina Truseq primers. Finally, libraries were analysed on an Illumina NextSeq500 using 75-bp pair-end sequencing.
In vitro transcription
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In vitro transcription is a laboratory technique used to produce RNA molecules from a DNA template. It involves the enzymatic synthesis of RNA using a DNA-dependent RNA polymerase enzyme. The core function of in vitro transcription is to generate specific RNA molecules for various applications, such as research, diagnostics, and therapeutics.
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5 protocols using in vitro transcription
1
Single-Cell Transcriptome Profiling of Ngn3-Cre-RFP Organoids
2
RNAi Depletion of ACER-1 and CRA-1 in C. elegans
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RNAi by microinjection was performed to deplete ACER-1. Double stranded RNA was produced by in vitro transcription (Ambion) and was injected into the gonads of young adult worms (12 h post-L4) at a concentration of 1 μg/μl. The acer-1(RNAi) worms examined consisted of young adults (24h post-L4) from the F1 generation. Single worm RT-PCR detection shows that acer-1 expression is only partially suppressed following RNAi depletion (S5B Fig. ). Feeding RNAi was used for depletion of CRA-1 as described in [50 (link)]. CRA-1 cDNA was cloned into the pL4440 feeding vector. Control RNAi was performed by feeding worms with HT115 bacteria carrying the empty pL4440 vector.
3
dsRNA Production and Depletion Assay
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The template for air-2 dsRNA production was PCR amplified from the cDNA clone yk483g8 with T7 and T7_T3 primers (Supplementary file 2 ). The templates for chk-1 and chk-2 dsRNA production were amplified from the Open Biosystems RNAi library (Fisher Scientific, Pittsburgh, PA) clones GHR-10020 and GHR-11002, respectively. Other templates for dsRNA production were amplified from genomic DNA with gene-specific primers that included 5′ T7 sequences (Supplementary file 2 ). In all cases, PCR products were gel purified, then reamplified with T7 primers. dsRNA was prepared by in vitro transcription (Ambion, Austin, TX). Young adult hermaphrodites were injected with dsRNA at concentrations of 2.5–5 mg/ml, then mated with him-8; mIs10 males at 20°C. Worms and embryos were fixed and stained 72 hr post injection for depletion of AIR-2, ATL-1, and SMC-1, 60 hr post injection for depletion of CHK-1 and CHK-2, and 48 hr post injection for depletion of SCC-1.
4
In Situ Hybridization and RT-PCR for LMF1 Gene
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In situ hybridization was carried out by Phylogeny Inc. as described [11 (link)]. 35S-UTP-labeled sense and anti-sense cRNA probes were generated by in vitro transcription (Ambion) from a T7 and T3 promoter-appended PCR product (primers T7-LMF1 and T3-LMF1 in Table 1 ) representing the 5’-terminal 889 bp of the LMF1 open reading frame. RNA and cDNA from adult tissues was prepared as described previously [12 (link)] and cDNA from embryonic tissues was obtained from OriGene (TissueScan, MDRT). Real-time PCR was performed using LMF1 (#4351372, Life Technologies) and GAPDH (#4352339E) TaqMan assays (Figure 1 B), or SybrGreen assays using primers for LMF1 (p6 and p7 in Table 1 ) and 36B4. For semi-quantitative RT-PCR analysis of the gene-trapped LMF1 allele, primers (p1-p5 in Table 1 ) spanning multiple exons were used as shown in Figure 2 A.
5
RNA FISH Probe Labeling and Detection
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RNA FISH probe labeling and RNA FISH procedures were performed as described previously [96 (link)]. Biotinylated single-strand LAT RNA probe was prepared by in vitro transcription (Ambion) using plasmid pSLAT-2 as a template (gift from S. Efstathiou, University of Cambridge, UK). Biotinylated LacZ probe was prepared from the pCMV-LacZ plasmid (Clontech) using the nick-translation procedure (Invitrogen). Frozen sections were treated as described for DNA FISH up to the antigen-unmasking step using solutions containing 2 mM of the RNAse inhibitor ribonucleoside vanadyl complex. The sections were pre-hybridized in 50% formamide/2 × SSC and hybridized overnight with 60 ng of RNA probe in a 50% formamide buffer at 65°C for LAT and 37°C for LacZ. Sections were washed in 50% formamide/2 × SSC at 65°C, and in 2 × SSC at room temperature. Detection was performed using streptavidin-HRP conjugate, followed by Tyramide Signal Amplification (TSA, Invitrogen) with an Alexa Fluor 350- or 488-conjugated substrate, according to the manufacturer’s guidelines. The DNA-FISH procedure was performed starting from the methanol/acetic acid post-fixation step.
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