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Sample GSM3814847 Query DataSets for GSM3814847
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
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
BioSample SAMN11843114
SRA SRX5891251

Supplementary file Size Download File type/resource 21.5 Mb (ftp)(http) BW 21.1 Mb (ftp)(http) BW
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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