Experiment / E1CC8N86WStandard STARR-seq

FACS-assisted whole-genome STARR-seq in Nipponbare rice protoplasts

Genome‐wide prediction of activating regulatory elements in rice by combining STARR‐seq with FACS

A whole-genome sheared Nipponbare genomic library was cloned into an episomal self-transcribing reporter and transfected into rice stem and sheath protoplasts. GFP-positive cells were isolated by FACS before ARE RNA/cDNA sequencing, with matched plasmid-input libraries in two independent replicates; the packaged table contains the authors' 1,818 final high-confidence genomic regulatory elements.

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Organism: NCBITaxon:4530
Taxonomy ID: NCBITaxon:4530
Biosample: UNMAPPED:rice_stem_sheath_protoplast
Reference genome: IRGSP1.0
Design focus: Region-focused
Region of interest: Unbiased whole-genome assay (*)

Perturbation & assay details

Basal / Untreated

FACS-assisted conventional STARR-seq using an episomal vector containing a minimal cauliflower mosaic virus 35S promoter, the first intron of castor bean catalase (Cat1), EGFP, a multiple cloning site, and a NOS terminator. Nipponbare genomic DNA was sheared mainly to 700–1500 bp, cloned into the reporter 3′ UTR, and delivered to stem/sheath protoplasts by PEG transfection. GFP-positive cells were sorted with a 488-nm FACS Aria III workflow; ARE RNA/cDNA and matched plasmid-input libraries were sequenced as paired-end 150-bp reads on Illumina HiSeq X Ten for R1 and R2. The authors compared ARE treatment with input using Bowtie2 mapping to IRGSP1.0 and MACS2 peak calling.

Processed data

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Column dictionary · 41 definitions

element_id: Stable genomic element identifier formed from the source chromosome and coordinate interval.
source_fasta_header: Original G1/G2 FASTA header from Data S2, including the authors' GC-content group label.
chromosome: Rice chromosome reported by the source workbook.
start: Start coordinate of the supplied element interval, preserved from Data S2.
end: End coordinate of the supplied element interval, preserved from Data S2.
element_length_bp: Length of the supplied element sequence in base pairs; equals end - start + 1 for these records.
sequence: Nucleotide sequence of the tested genomic insert from the G1 or G2 sheet.
gc_content_percent: GC percentage calculated from the supplied element sequence.
gc_group: Authors' sequence cluster: G1 or G2, based on GC-content pattern.
gc_subgroup: Authors' G1 positional subgroup, G1a or G1b; blank for G2.
author_retained_high_confidence: TRUE for records included in the authors' final high-confidence G1/G2 element set after FE > 20 screening.
replicate_support_count: Number of independent source peak sheets (R1 and R2) with an overlapping call for the element.
replicate_1_peak_present: TRUE when the element interval overlaps a Replicate 1 source peak call; otherwise FALSE.
replicate_1_peak_start: Start coordinate of the selected overlapping Replicate 1 peak call; blank when not detected.
replicate_1_peak_end: End coordinate of the selected overlapping Replicate 1 peak call; blank when not detected.
replicate_1_peak_length_bp: Length of the selected Replicate 1 source peak call.
replicate_1_abs_summit: Absolute summit coordinate of the selected Replicate 1 MACS2 peak.
replicate_1_pileup: MACS2 pileup value for the selected Replicate 1 peak.
replicate_1_log10_pvalue: MACS2 -log10(p-value) for the selected Replicate 1 peak.
replicate_1_fold_enrichment: MACS2 fold enrichment over input for the selected Replicate 1 peak.
replicate_1_log2_fold_enrichment: Derived log2 of Replicate 1 fold enrichment over input.
replicate_1_log10_qvalue: MACS2 -log10(q-value) for the selected Replicate 1 peak.
replicate_1_source_cutoff_pass: TRUE when the selected Replicate 1 source peak has fold enrichment > 20, the paper's final screening cutoff.
replicate_1_match_overlap_bp: Number of base pairs overlapping between the element interval and the selected Replicate 1 source peak.
replicate_1_match_overlap_fraction: Overlap divided by the shorter of the element and selected Replicate 1 peak intervals.
replicate_1_match_count: Number of Replicate 1 source peak calls overlapping the element interval before selecting the best overlap.
replicate_2_peak_present: TRUE when the element interval overlaps a Replicate 2 source peak call; otherwise FALSE.
replicate_2_peak_start: Start coordinate of the selected overlapping Replicate 2 peak call; blank when not detected.
replicate_2_peak_end: End coordinate of the selected overlapping Replicate 2 peak call; blank when not detected.
replicate_2_peak_length_bp: Length of the selected Replicate 2 source peak call.
replicate_2_abs_summit: Absolute summit coordinate of the selected Replicate 2 MACS2 peak.
replicate_2_pileup: MACS2 pileup value for the selected Replicate 2 peak.
replicate_2_log10_pvalue: MACS2 -log10(p-value) for the selected Replicate 2 peak.
replicate_2_fold_enrichment: MACS2 fold enrichment over input for the selected Replicate 2 peak.
replicate_2_log2_fold_enrichment: Derived log2 of Replicate 2 fold enrichment over input.
replicate_2_log10_qvalue: MACS2 -log10(q-value) for the selected Replicate 2 peak.
replicate_2_source_cutoff_pass: TRUE when the selected Replicate 2 source peak has fold enrichment > 20, the paper's final screening cutoff.
replicate_2_match_overlap_bp: Number of base pairs overlapping between the element interval and the selected Replicate 2 source peak.
replicate_2_match_overlap_fraction: Overlap divided by the shorter of the element and selected Replicate 2 peak intervals.
replicate_2_match_count: Number of Replicate 2 source peak calls overlapping the element interval before selecting the best overlap.
qc_status: Packaging QC result; PASS indicates a valid nucleotide sequence and a coordinate-length consistency check.

Quality control

The authors aligned reads to the Nipponbare IRGSP1.0 genome with Bowtie2, retained mapped reads passing SAMtools -q 30 -f 2 -F 264, and called ARE-versus-input transcription peaks with MACS2 (-g 4.4e8 -q 0.01 --keep-dup all). They retained duplicates because the library used minimal PCR amplification. For packaging, the authors' final G1/G2 sequence set was used as the element universe; this is the 1,818-element set produced after the reported FE > 20 screening step, rather than all 29,172/26,916 source peak rows. Source calls were joined to this universe by genomic interval overlap, and elements were not discarded when a particular replicate had no overlapping call. Every retained sequence contains only A/C/G/T/N and has a length equal to its supplied inclusive coordinate interval; all 1,818 rows passed package QC. The table reports source peak metrics for each replicate where an overlapping call exists; FASTQ files were not downloaded, so no additional read-level QC was performed.

Curation notes

This is one MPRA experiment with two independent sequencing replicates, not two different biological conditions. The source workbook's Replicate 1 and Replicate 2 sheets contain 29,172 and 26,916 MACS2 calls, respectively; the G1 and G2 sheets contain the authors' 1,818 final sequence records used for downstream analysis. The interval-overlap join reproduces the paper's reported support split of 1,169 elements in both replicates, 372 in R1 only, and 277 in R2 only. The author-retained flag refers to inclusion in the G1/G2 sequence universe; the per-replicate source_cutoff_pass fields instead evaluate the selected joined source peak and can differ when independently represented peak boundaries overlap. G1/G2 and G1a/G1b are sequence-clustering labels, not fluorescence bins or allele labels. The paper's traditional STARR-seq validation constructs and CRISPR disruption assays were not made separate experiments because no comparable genome-wide quantitative MPRA table was supplied for those assays. Rice protoplasts have no resolved Cell Ontology or Cellosaurus identifier in the requested vocabulary, so the biosample is explicitly marked UNMAPPED.

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