|
| Status |
Public on Dec 11, 2019 |
| Title |
s03_WT_Cold_3h_rep1 |
| Sample type |
SRA |
| |
|
| Source name |
PlaNET-Seq of wild type exposed to 4⁰C for 3 hours
|
| Organism |
Arabidopsis thaliana |
| Characteristics |
ecotype background: Columbia (Col-0)
genotype/variation: Wild type
age: 10 day
tissue: Whole seedlings
treatment: 4⁰C for 3 hours
molecule subtype: Nascent RNA
protocol: PlaNET-Seq
|
| Treatment protocol |
For inhibition of splicing, seedlings were grown on filter paper covered 1/2 MS + 1% sucrose for 10 days then transferred to either DMSO or 5 µM pladienolide B (Santa Cruz) containing plates for 6 hours. For cold treatment, 10-day old seedlings grown were transferred to 4°C and approximately 25 µE m-2 s-1 for either 3 hours or 12 hours.
|
| Growth protocol |
PlaNET-Seq: Arabidopsis seedlings were grown on plates (1/2 Murashige and Skoog medium, 1% sucrose) with a 16h light/8h dark cycle at 22°C/18°C. Light intensity during day hours was approximately 100 µE m-2 s-1. TSS-Seq: as described previously in Nielsen et al., 2019 (PMID 30707695).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
PlaNET-Seq: Nuclei were isolated from 3 grams of seedlings. Chromatin was solubilized by DNase I treatment. Nascent RNA fraction was enriched by immunoprecipitation of FLAG-tagged RNAPII elongation complexes using anti-FLAG antibody (F3165, Sigma-Aldrich) coupled to Dynabeads Protein G. Finally, RNAPII complexes were eluted from Dynabeads by 3xFLAG peptide, and the nascent RNA was purified using miRNeasy kit (QIAgen). TSS-Seq: as described previously in Nielsen et al., 2019 (PMID 30707695).
PlaNET-Seq: NGS libraries were constructed from nascent RNA using Small RNA-Seq Kit v3 (Bioo Scientific). The original protocol was modified to incorporate alkaline RNA fragmentation step after 3' adapter ligation. TSS-Seq: as described previously in Nielsen et al., 2019 (PMID 30707695).
|
| |
|
| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 4000 |
| |
|
| Description |
s03_WT_Cold_3h_biorep1
|
| Data processing |
Illumina Casava v1.7 software was used for basecalling.
PlaNET-Seq: 1) Trim 4bp UMI barcodes from 5' ends of both R1 and R2 reads (UMI-Tools extract v0.5.3); 2) Align R2 reads to TAIR10 (STAR v2.5.2b; --outSAMmultNmax 1 --alignEndsType Extend5pOfRead1 --clip3pAdapterSeq GATCGTCGGACT); 3) Sort BAM files (Samtools v1.3.1); 4) Remove PCR duplicates (UMI-Tools dedup); 5) Remove reads aligned to rRNA, tRNA, snRNA or snoRNA loci from Araport11 (BEDTools v2.17.0); 6) Remove reads with MAPQ < 10 (Samtools v1.3.1); 7) Import BAM files into R environment v3.5.1 (GenomicAlignments_1.18.1, GenomicRanges_1.34.0); 8) Flip the strand orientation; 9) Skip all split reads; 10) Skip reads with 3' end overlapping known splice sites (combined from TxDb.Athaliana.BioMart.plantsmart28_3.2.2 and Araport11); 11) Convert reads to genomic coverage (separately for + and - strands); 12) Export as BigWig files (rtracklayer_1.42.2). TSS-Seq: data processing was described previously in Nielsen et al., 2019 (PMID 30707695).
Genome_build: TAIR10
Supplementary_files_format_and_content: BigWig files show strand-specific sequencing coverage with single base resolution (no signal smoothing, transformation or normalization were applied). For TSS-Seq, only positions with coverage above 2 tags were reported.
|
| |
|
| Submission date |
May 23, 2019 |
| Last update date |
Dec 11, 2019 |
| Contact name |
Maxim Ivanov |
| E-mail(s) |
maxim.ivanov@plen.ku.dk
|
| Organization name |
University of Copenhagen
|
| Department |
Dept. of Plant and Environmental Sciences
|
| Street address |
Thorvaldsensvej 40
|
| City |
Frederiksberg C |
| ZIP/Postal code |
1871 |
| Country |
Denmark |
| |
|
| Platform ID |
GPL21785 |
| Series (1) |
| GSE131733 |
Transient genome-wide adaptations of nascent RNAPII transcription triggered by low temperature |
|
| Relations |
| BioSample |
SAMN11843114 |
| SRA |
SRX5891251 |
