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| Status |
Public on Apr 06, 2022 |
| Title |
CS_seedling_GRO_seq_v2_rep3 |
| Sample type |
SRA |
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| Source name |
Triticum aestivum 12-days-old seedlings
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| Organism |
Triticum aestivum |
| Characteristics |
tissue: 12-days-old seedlings
cultivar: Chinses Spring
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| Growth protocol |
The bread wheat (Triticum aestivum) cultivar “Chinese Spring” were grown under long day condition.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Leaf tissue of 12-day-old seedlings were collected by flash freezing with liquid nitrogen and followed by nuclei isolation.
For GRO-seq, nuclei were isolated by the Percoll gradient procedure as previously described except that the concentration of Triton X-100 was reduced to 0.5%. In vitro run-on, and nascent RNA purification were performed as previously reported20. The obtained nascent RNA was treated with T4 PNK (NEB), followed by TRIzol purification and subject to small RNA library construction using NEXTflex™ Small RNA-Seq Kit v3 (PerkinElmer) according to the manufacturer manual. The cDNA libraries were sequenced on the Illumina NovaSeq platform.For the second time (v2_rep1, v2_rep2, and v2_rep3), to obtain a deeper sequencing coverage at a reasonable sequencing cost, we added a rRNA removal step after nucleus RNA isolation and before the affinity purification of nascent RNA using riboPOOL kit (siTOOLs BIOTECH) . For pNET-seq, nuclei were isolated by a lysis-and-wash procedure as previously described except that the concentration of Triton X-100 was reduced to 0.5%. The RNA Pol II associated RNA were immunoprecipitated using Pol II antibody (Abcam, ab817) and followed by small RNA cDNA library construction. The size selection of nascent RNA was omitted. The cDNA libraries were sequenced on the Illumina NovaSeq platform.
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 3000 |
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| Data processing |
Library strategy: GRO-seq
Sequencing reads were cleaned with Trim Galore (version 0.6.4) and cutadapt (version 1.16) programs to remove sequencing adapters, low quality bases (< 20), short reads and the 4 bases in 5’end and 3’end of R1 and R2 which were randomly introduced.
Clean reads were aligned to the International Wheat Genome Sequencing Consortium (IWGSC) reference sequence (version 1.0) with the bowtie2 (version 2.3.5.1) program and only keep the reads with one best unique alignment. We also used SortMeRNA (version 2.1b) program to remove the reads originating from chloroplast, mitochondria and rRNA.
For GRO-seq, the 5′ coordinate of R1 reads was considered the position of the engaged polymerase for future analyses. Each site density of GRO-seq was then calculated in plus and minus strands and normalized by the count of unique mapped reads. For pNET-seq, the 5′ coordinate of R2 reads with the directionality indicated by R1 reads was considered the position of the engaged polymerase.
Genome_build: IWGSC RefSeq v1.0
Supplementary_files_format_and_content: GRO-seq and pNET-seq signal in each site were provided in bedgraph file.
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| Submission date |
Jan 18, 2022 |
| Last update date |
Apr 08, 2022 |
| Contact name |
yijing zhang |
| E-mail(s) |
zhangyijing@fudan.edu.cn
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| Organization name |
Fudan University
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| Department |
Biochemistry
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| Lab |
Functional Epigenomics Group
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| Street address |
2005 Songhu Road
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| City |
shanghai |
| ZIP/Postal code |
200438 |
| Country |
China |
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| Platform ID |
GPL24354 |
| Series (2) |
| GSE178276 |
Nascent transcriptomic landscape in bread wheat detected Pol II-dependent enhancer transcription |
| GSE178372 |
Distinct chromatin signatures and transcriptional landscape of functional genetic elements in allohexaploid wheat |
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| Relations |
| BioSample |
SAMN25067986 |
| SRA |
SRX13823256 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5823400_CS_seedling_GRO_seq_v2_rep3_minus.bedgraph.gz |
26.6 Mb |
(ftp)(http) |
BEDGRAPH |
| GSM5823400_CS_seedling_GRO_seq_v2_rep3_plus.bedgraph.gz |
26.7 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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