|
| Status |
Public on Feb 14, 2020 |
| Title |
s03_nrpb2-Y732F_plaNETseq_biorep1 |
| Sample type |
SRA |
| |
|
| Source name |
Whole seedlings
|
| Organism |
Arabidopsis thaliana |
| Characteristics |
tissue: Whole seedlings
ecotype: Columbia (Col-0)
genotype: nrpb2-Y732F
age: 10 day
|
| Growth protocol |
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.
|
| 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). RNA-Seq: total RNA was isolated from seedlings using QIAgen Plant RNeasy kit.
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. RNA-Seq: polyA-enriched libraries were constructed using Illumina TruSeq RNA Sample Prep Kit v2.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
| |
|
| Description |
library strategy: PlaNET-Seq
Nascent RNA
PlaNET-Seq of mutant seedlings with accelerated transcription speed (replicate 1)
|
| 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). RNA-Seq: 1) Trim adapters using Trim Galore v0.4.3 (--paired --illumina); 2) Align to TAIR10 using STAR v2.5.2b (--outSAMmultNmax 1 --alignEndsType Local --outSAMtype BAM Unsorted); 3) Sort BAM files and remove reads with MAPQ < 10 using Samtools v1.3.1; 4) Convert BAM files to Bedgraph using BEDtools genomecov v2.26.0 (-bg -split); 5) Convert Bedgraph files to BigWig using kentUtils bedGraphToBigWig v4.
Genome_build: TAIR10
Supplementary_files_format_and_content: BigWig files show sequencing coverage with single base resolution (no signal smoothing, transformation or normalization were applied). PlaNET-Seq BigWigs are strand-specific, RNA-Seq BigWigs are unstranded.
|
| |
|
| Submission date |
Jun 21, 2019 |
| Last update date |
Feb 14, 2020 |
| 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) |
| GSE133143 |
Organismal Benefits of Transcription Speed Control at Gene Boundaries |
|
| Relations |
| BioSample |
SAMN12107340 |
| SRA |
SRX6102431 |
