Related files and analysis scripts for "An integrated experiment-to-analysis workflow to simply and affordably achieve precise mapping of transcription start sites in plants"

1, In vitro transcription (IVT) experiments datasetThis dataset contains raw sequencing reads and processed data from an IVTexperiment designed to evaluate MMLV reverse transcriptase slippage at the 5' cap structure during template-switching. A DNA fragment encoding EGFP under the T7 promoter was amplified by PCR and used as template for capped RNA synthesis with the EasyCap T7 Co‑transcription Kit (Vazyme, DD4203). Template‑switching reverse transcription was then performed using a TSO containing the rGrGrG motif under standard Cap‑STRIPE conditions, and the resulting cDNA was converted into an Illumina‑compatible sequencing library. The library was sequenced on an Illumina NovaSeq X Plus platform to generate 150‑bp paired‑end reads. IVT-1 to IVT-3: three independent IVT replicates; _1 and _2: paired-end read 1 and read 2 files. 2, Corrected genome annotation filesThis repository also includes experimentally corrected transcription start site (TSS) and transcription end site (TES) annotations for maize (Zea mays), rice (Oryza sativa), and foxtail millet (Setaria italica). These annotations were derived by refining the following reference genome annotations—Zea_mays.Zm-B73-REFERENCE-NAM-5.0.62, Oryza_sativa.IRGSP-1.0.62, and Setaria_italica_v2.0.62—using Cap‑STRIPE in combination with QUIC‑seq (Teng et al., 2025). The corrected GTF files provide high‑precision, experimentally validated boundaries suitable for regulatory genomics, evolutionary analysis, and generative modeling of gene expression. Maize: Zm_CapSTRIPE_QUICseq_corrected.gtf; Rice: Os_CapSTRIPE_QUICseq_corrected.gtf; Foxtail millet: Si_CapSTRIPE_QUICseq_corrected.gtf. 3, De novo transposable element annotations filesde novo transposable element annotations to identify TE‑associated promoters by EDTA (version 2.3) Maize: Zea_mays.Zm-B73-REFERENCE-NAM-5.0.dna.toplevel.fa.mod.EDTA.TEanno.gff3; Rice: Oryza_sativa.IRGSP-1.0.dna.toplevel.fa.mod.EDTA.TEanno.gff3; Foxtail millet: Setaria_italica.Setaria_italica_v2.0.dna.toplevel.fa.mod.EDTA.TEanno.gff3. 4, Blastx resultFor intergenic CCs that did not overlap TEs or novel transcripts, flanking sequences (−500 bp to +3500 bp around the dominant TSS) were extracted using BSgenome packages and searched with BLASTX (version 2.17.0) against a combined protein database of eight grass species (Zea mays, Oryza sativa, Setaria italica, Setaria viridis, Sorghum bicolor, Triticum aestivum, Hordeum vulgare, Brachypodium distachyon) and Arabidopsis thaliana. Maize: Zm_blastx_results.tsv; Rice: Os_blastx_results.tsv; Foxtail millet: Si_blastx_results.tsv. 5, Candidate novel gene annotation filesRNA‑seq data were processed with STAR and StringTie to generate a candidate novel gene annotation Maize: Zm_novel_intergenic_transcripts_u.gtf; Rice: Os_novel_intergenic_transcripts_u.gtff; Foxtail millet: Si_novel_intergenic_transcripts_u.gtf. 6, Analyze code filesQUICseq_analysis_1.sh (custom_umi_extract_fast.py, extract_pas_and_dedup.py) and QUICseq_analysis_2_QuantifyPolyA.R are the analysis scripts for QUIC-seq.antisense_promoters.R, divergent_promoters.R, intergenic_promoters.R, and multi_promoters.R are used to analyze the CCs result files obtained from Cap-STRIPE.