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Sample GSM2193125

Query DataSets for GSM2193125

Status

Public on Oct 06, 2016

Title

GRO-seq rep2

Sample type

SRA

Source name

6 day seedlings

Organism

Arabidopsis thaliana

Characteristics

library strategy: nascent RNA

Growth protocol

A. thaliana accession Columbia (Col-0) was used as the wild type genetic background for experiments. All seeds were sterilized with chlorine gas by mixing 100 ml of bleach and 5 ml of concentrated HCl then grown on plates containing half Linsmaier and Skoog Medium (Caisson Laboratories). Plates were placed at 4C for 3 days for vernalization then placed in growth chambers with 24 hours light and 22¡C for 6 days before tissue collection.

Extracted molecule

total RNA

Extraction protocol

For RNA-seq, total RNA was extracted from seedlings frozen in liquid nitrogen using RNeasy Plant Mini Kit (Qiagen). 10µg of total RNA was used for extraction of mRNA with the Poly(A)Purist MAG Kit (Ambion) according to manufacturer’s instructions. For GRO-seq/5'GRO-seq, approximately 20 grams of 6-day-old seedlings were homogenized with an OMNI International General Laboratory Homogenizer in ice cold grinding buffer (300 mM sucrose, 20 mM Tris, pH 8.0, 5 mM MgCl2, 5 mM KCl, 0.2% Triton X-100, 5 mM β-mercaptoethanol, 35% glycerol) at 4°C on low speed. Samples were filtered twice through a 250 micron nylon mesh and then miracloth before being split into 50ml conical tubes and spun for 10 minutes at 5000g. Supernatant was discarded, and the pellets were resuspended in grinding buffer using a Kimble loose dounce homogenizer (Fisher Scientific). Wash step was repeated twice. Nuclei were resuspended in freezing buffer (50 mM Tris, pH 8.0, 5 mM MgCl2, 20% glycerol, 5 mM β-mercaptoethanol) and frozen in liquid N2. Approximately 5x106 nuclei were used for run-ons at room temperature for 5’ and CTP concentration was limited to 20 nM. After run-on, samples were treated with RQ1 DNaseI (Promega) for 20 minutes at 37°C followed by Trizol LS (Fisher Scientific) extraction as described by the manufacturer. Samples were then treated with Terminator 5’-Phosphate-Dependent Exonuclease (Epicentre) for 1 hour at 30°C. RNA fragmentation was carried out with Fragmentation Reagents kit (Ambion) for 11’ at 70°C. Treatments were used to deplete ribosomal and chloroplast RNA. Nascent RNA was enriched by double pull down using BrdU antibody beads (Santa Cruz Biotechnology) for 80’ at 4C, washed three times with GRO-Binding buffer (0.25x SSPE, 0.05% Tween, 37.5 mM NaCl , 1 mM EDTA) then extracted with Trizol LS (Fisher Scientific).
Isolated mRNA was fragmented with Fragmentation Reagents (Ambion) for 14’. Fragmented RNA was incubated with T4 polynucleotide kinase (New England Biolabs) in low pH buffer (0.1 M MES, 10 mM MgCl2, 10 mM mercaptoethanol, 300 mM NaCl - pH 5.6) for 3’ repair, then T4PNK buffer for 5’ phosphorylation. For 5'GRO-seq, de-capping using TAP was replaced with RppH (New England Biolabs) and NEB Next Small RNA Library Prep Set (New England Biolabs) was used for library preparation. De-capping was performed in 3’ Ligation buffer at 37°C in the absence of 3’ adapter. 3’Adapter ligation was performed at 20°C instead of 25°C so RppH is inactive. The Small RNA Library Prep Set was (New England Biolabs) was used for final library preparation of short RNA fragments from each method.
Strand specific polyA RNA-seq, GRO-seq, and 5'GRO-seq

Library strategy

OTHER

Library source

transcriptomic

Library selection

other

Instrument model

Illumina NextSeq 500

Description

GRO-seq in 6 day seedlings (rep 2)

Data processing

Library strategy: GRO-Seq
Adapter sequences were trimmed from the 3’ ends of all RNA-seq, GRO-seq, and 5’GRO-seq reads and the reads were then aligned to the TAIR10 arabidopsis genome using STAR. Only reads that aligned to the genome at a single unique location were considered for downstream analysis.
Transcript discovery and annotation were performed using routines in HOMER (v4.8). Transcription units were identified directly from GRO-seq data using the HOMER program findPeaks (-style groseq with default parameters). Transcription start sites (TSS) were found using findPeaks (-style tss) with the 5'GRO-seq data using using normal GRO-seq as a control. TSS discovered by 5’GRO-seq were then assigned to GRO-seq transcripts if they were found within 1 kb of the 5’ end of the GRO-seq transcript. In cases where multiple TSS could be assigned to the same transcript, the TSS with the highest read density was used. Only de novo identified transcripts that were assigned a valid 5’GRO-seq TSS were considered. Transcripts were annotated into different classes as follows: First, transcripts that strand-specifically overlap any known transcript (TAIR10) were assigned the accession number/annotation of the known transcript. Next transcripts that overlapped any known transcript on the opposite strand are assigned as ‘antisense transcripts’. Next, transcripts found upstream of an annotated TSS in the opposite strand of the annotated genes were assigned as ‘promoter-antisense transcripts’. Transcripts failing to meet any of these critierion were assigned as ‘novel’ transcripts.
Genome_build: TAIR10
Supplementary_files_format_and_content: tab delimited text

Submission date

Jun 08, 2016

Last update date

May 15, 2019

Contact name

Christopher Benner

E-mail(s)

cbenner@ucsd.edu

Organization name

University of California, San Diego (UCSD)

Department

Medicine