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Sample GSM3814850 Query DataSets for GSM3814850
Status Public on Dec 11, 2019
Title s06_WT_Cold_12h_rep2
Sample type SRA
 
Source name PlaNET-Seq of wild type exposed to 4⁰C for 12 hours
Organism Arabidopsis thaliana
Characteristics ecotype background: Columbia (Col-0)
genotype/variation: Wild type
age: 10 day
tissue: Whole seedlings
treatment: 4⁰C for 12 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 s06_WT_Cold_12h_biorep2
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 SAMN11843111
SRA SRX5891254

Supplementary file Size Download File type/resource
GSM3814850_s06_WT_Cold_12h_biorep2_Minus.bw 21.7 Mb (ftp)(http) BW
GSM3814850_s06_WT_Cold_12h_biorep2_Plus.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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