Yeast RNA was prepared by extraction with hot acidic phenol (19 ). Total RNA was extracted, and 4tU-containing mRNA was biotinylated and isolated as previously described (20 (link),21 (link)). To obtain high quality RNA, samples were further subjected to the clean-up protocol with the RNeasy Mini Kit (Qiagene, Cat. #74104), and quality was checked with gel electrophoresis. Nascent RNA was then biotinylated as described (20 (link),21 (link)), using 400 μl (∼40 μg) total RNA and 4 μg MTSEA biotin-XX (Biotium) and purified by MyOne Streptavidin C1 Dynabeads (Invitrogen). The isolated RNA was further purified and concentrated using Qiagen miRNeasy columns (#217084), according to the manufacturer's protocol. The isolated RNA was then prepared for sequencing using the Ovation SoLo or Ovation Universal RNA-seq System kits (Tecan), according to the manufacturer's instructions. Libraries were sequenced on the Illumina NextSeq high output platform using single reads 75 in Institute of Molecular Biology of Academia Sinica
Ovation solo
Manufactured by Tecan
The Ovation SoLo is a compact and versatile laboratory instrument designed for automated liquid handling. It features a low-profile design and intuitive user interface to streamline various sample preparation and liquid handling workflows in research and analytical laboratories.
Automatically generated - may contain errors
Lab products found in correlation
Ovation universal rna seq system kits, by Tecan (3 mentions)
Hiseq 2500, by Illumina (2 mentions)
2100 bioanalyzer, by Agilent Technologies (2 mentions)
Novaseq 6000, by Illumina (2 mentions)
Nextseq high output platform, by Illumina (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Mirneasy column, by Qiagen (1 mentions)
Dynabeads myone streptavidin c1, by Thermo Fisher Scientific (1 mentions)
Nextseq 2000, by Illumina (1 mentions)
Hiseq 2500 platform, by Illumina (1 mentions)
6 protocols using ovation solo
1
Yeast RNA Extraction and 4tU Labeling
2
RNA-seq Library Preparation from 4-ThioU Labeled Transcripts
3
RNA Isolation and Sequencing Protocol
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Newly synthesized RNA isolated via 4-thioU labeling and purification was prepared for sequencing using the Ovation SoLo or Ovation Universal RNA-seq System kits (Tecan) according to the manufacturer’s instructions and 1 ng (SoLo) or 50 ng (Universal) input RNA. Libraries were sequenced on the Illumina HiSeq2500 platform using 25 bp paired-ends at the Fred Hutchinson Genomics Shared Resources facility.
4
RNA-Seq Analysis of Mouse Transcriptome
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RNA quality was checked using a Bioanalyzer 2100 (Agilent technologies). Indexed cDNA libraries were obtained using the Ovation Solo (NuGen) or the NEBNext RNA-Seq Systems following manufacturer recommendations. The multiplexed libraries were loaded onto a NovaSeq 6000 (Illumina) using an S2 flow cell and sequences were produced using a 200 Cycle Kit. Paired-end reads were mapped against the mouse reference genome GRCm38 using STAR software to generate read alignments for each sample. Annotations Mus_musculus.GRCm38.90.gtf were obtained from ftp.Ensembl.org. After transcripts assembling, gene-level counts were obtained using HTSeq. Differentially expressed genes were identified with EdgeR method and further analyzed using GSEA MolSig (Broad Institute) [45 (link)]. Heatmaps were generated using Heatmapper [46 (link)] and common signature genes were identified using Venny 2.0 [47 ].
5
RNA-seq analysis of gastric eosinophils
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RNA from FACS-sorted gastric eosinophils was isolated using the Arcturus PicoPure RNA Isolation kit (Applied Biosystems, ThermoFisher Scientific). RNA quality was assessed by the Agilent Tapestation followed by library preparation using the NuGEN Ovation SoLo protocol. Sequencing was subsequently performed on the Illumina HiSeq 2500 instrument. RNA-seq reads were quality-checked with fastqc, which computes various quality metrics for the raw reads. RNA-seq reads were mapped using the STAR aligner to the mouse reference genome build GRCm38. We included the Ensembl transcript annotations in the mapping index. Alignments were counted according using the featureCounts function in the Rsubread Bioconductor package. Statistical analysis of differential expression was conducted with the DESeq2 package. All RNA seq data generated within this study are publicly accessible at the Gene Expression Omnibus (GEO) repository (no. GSE105143 ).
6
RNA-Seq analysis of Lgr5+ stem cell
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RNA quality was checked using a Bioanalyzer 2100 (Agilent technologies). Indexed cDNA libraries were obtained using the Ovation SoLo (NuGen) or the NEBNext RNA‐Seq Systems following manufacturer recommendation. The multiplexed libraries were loaded on a NovaSeq 6000 (Illumina) using a S2 flow cell, and sequences were produced using a 200 Cycle Kit. Paired‐end reads were mapped against the mouse reference genome GRCm38 using STAR software to generate read alignments for each sample. Annotations Mus_musculus.GRCm38.90.gtf were obtained from ftp.Ensembl.org . After transcripts assembling, gene‐level counts were obtained using HTSeq. Genes differentially expressed were identified with EdgeR method and further analyzed using GSEA MolSig (Broad Institute) 46 . Gene pattern was used to compare by pre‐ranked GSEA E16.5 Lgr5 KO versus HE; Adult HE versus E18.5 HE, or Adult Lgr5 KO versus HE transcriptomes with gene datasets 47 . Gene Ontology was used to identify biological processes 48 .
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