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Sample GSM7078149

Query DataSets for GSM7078149

Status

Public on Jul 05, 2024

Title

Ath RPFs, 700 U RNaseI, replicate2

Sample type

SRA

Source name

14-day old seedlings

Organism

Arabidopsis thaliana

Characteristics

tissue: 14-day old seedlings
nuclease treatment: 700 Units RNaseI
rrna depletion: no
genotype: Wild type
treatment: RPFs generated using 700 U RNaseI
molecule type: ribosome protected fragment

Treatment protocol

Growth protocol

The tissue used for the comparison of RNase I and MNase digestion, was derived from 8-day old Arabidopsis seedlings (Col-0) grown on ½ Murashige and Skoog medium (Murashige and Skoog, 1962) with 6.8% agar and 1% sucrose, grown at 100 µmol m-2s-1 for 16h/8h light/dark cycles at 20 °C. The tissue used for refining RNase I treatment and rRNA depletion were derived from 14-day old Arabidopsis seedlings (Col-0), grown on ½ Murashige and Skoog media with 1% agar, grown at 100 µmol m-2s-1 for 12h/12h light/dark cycles at 20 °C. Nicotiana tabacum tissue was derived from leaves harvested from 28-day old plants grown on soil at 350 µmol m-2s-1 in 16h/8h light/dark cycles at 12 °C.

Extracted molecule

other

Extraction protocol

RPF isolation: RPFs were isolated as previously described (Trösch et al., 2018) with the following modifications. The units (U) of ribonuclease used in this study are normalized to one mL of plant lysate, derived from 100 mg of plant fresh weight. 300 mg frozen plant tissue was homogenized in liquid nitrogen with a mortar and pestle followed by the addition of 3 mL ribosome extraction buffer (0.2 M sucrose, 0.2 M KCl, 40 mM Tris-OAc pH 8.0, 10 mM MgCl2, 10 mM 2-Mercaptoethanol, 2% (v/v) polyoxyethylene (10) tridecyl ether, 1% (v/v) Triton X-100, 100 μg/mL chloramphenicol, 100 μg/mL cycloheximide). After brief mixing, a 0.5 mL aliquot of the lysate was flash frozen and stored at -80 °C for later total RNA extraction using TRIzol reagent (ThermoFisher cat# 15596026). This total RNA can be used for standard RNA-seq experiments. This step is especially important if the calculation of translation efficiencies is desired (i.e., the normalization of Ribo-seq data to RNA-seq data). The remaining lysate was filtered through glass wool, followed by centrifugation for 10 min at 15,000 x g at 4 °C to remove cell debris. 2.5 mL of the clarified lysate were incubated at specified concentrations of RNase1 or MNase for 1 h at room temperature with gentle rotation to degrade mRNA regions that are not covered and protected by translating ribosomes. Since Ca2+ is a known cofactor of MNase, samples digested with MNase include 5 mM CaCl2. The ribonuclease-treated lysate contains mainly monosomes which were loaded onto a 2 mL sucrose cushion (30% (w/v) sucrose, 40 mM Tris-Acetate pH 8.0, 100 mM KCl, 15 mM MgCl2, 0.1 mg/mL chloramphenicol, 0.1 mg/mL cycloheximide, 0.2% β-MeEtOH) and centrifuged (OptimaTM L-80 XP Ultracentrifuge - Beckman Coulter) for 1.5 h at 303,800 x g at 4 °C. The supernatant was then carefully aspirated and the pelleted monosomes resuspended in 0.5 mL footprint isolation buffer (10 mM Tris pH 8.0, 1 mM EDTA pH 8.0, 100 mM NaCl, 1% (w/v) SDS, 0.1 M EGTA pH 8.0). The RNA was immediately extracted from this pellet using 0.5 mL TRIzol reagent. Next RPFs were size-selected through electrophoresis on a 12% denaturing polyacrylamide gel (19:1, acrylamide:bisacrylamide) prepared in 1x TBE buffer (89 mM Tris, 89 mM Boric Acid, 2mM EDTA pH 8.0) containing 8 M urea. To this end, 25 μg RNA were resuspended in 40 μL of ribosome footprint loading buffer (90% (v/v) deionized formamide, 20 mM Tris-HCl pH 7.5, 20 mM EDTA pH 8.0, 0.04% (w/v) bromophenol blue, and 0.04% (w/v) xylene cyanol) and denatured for 10 min at 70 °C. The gel was run in 1x TBE buffer with a constant power of 30 W at constant temperature of 12 °C (achieved by a cooling unit). Co-migrating pre-stained RNA ladder (Biodynamics Laboratory cat# DM253) was used to visualize the regions of the gel to excise RPFs. All RPFs that were not rRNA-depleted were size-selected between 20-50 nt. All rRNA-depleted RPFs were size-selected between 20-35 nt. RNA was eluted from the excised gel piece in 4 mL TESS (10 mM Tris pH 8.0, 1 mM EDTA pH 8.0, 0.1 M NaCl, 0.2% (w/v) SDS) by overnight incubation at 4 °C with gentle rotation. Eluted RNA was isolated with 4 mL of phenol:chloroform:isoamyl alcohol (25:24:1), followed by overnight ethanol precipitation at -20 °C. To increase purity and further narrow the volume, the RNA pellet was resuspended in 0.1 M NaCl (500 μL) and subjected to a second round of phenol:chloroform:isoamyl alcohol (25:24:1) extraction, followed by two washes with chloroform:isoamylalcohol (24:1) and overnight ethanol precipitation at -20 °C. The received RPF pellet was washed twice with 75% ethanol and resuspended in 20 μL RNase-free water.
Library preparation: T4 polynucleotide kinase (PNK; ThermoFisher, cat#EK0031) reactions were carried out in 18 µL volume with 100 ng of RPFs without ATP for 10 min at 37 °C to promote the 3’-dephosphorylation reaction. Next, 2 µL ATP (10 mM) was added, followed by another incubation for 30 min at 37 °C. PNK was inactivated by heating to 65 °C for 20 min. 20 ng RPFs were directly used as input for the NEXTflex small RNA-seq kit v3 (Perkin Elmer, cat# NOVA-5132-06) according to manufacturer’s instructions. The final libraries were PCR amplified using 15-17 PCR cycles.

Library strategy

OTHER

Library source

other

Library selection

other

Instrument model

Illumina NextSeq 500

Description

Ath_RNaseI_700U_B
counts_over_CDS-Arabidopsis.xlsx
library_stats.xlsx

Data processing

library strategy: Ribosome Profiling (Ribo-seq)
Arabidopsis:The Arabidopsis GTF gene annotations (v45) and TAIR10 genome were downloaded from ensembl (https://plants.ensembl.org/index.html). Cutadapt (Martin, 2011) was used to remove adapters and UMIs. Ribo-seq contaminants are generally defined as rRNA, tRNA and snoRNA. These sequences were extracted from the gene annotation file, and converted to fasta format using bedtools (Quinlan and Hall, 2010). Importantly, the nuclear 25S and 5S are missing from the Arabidopsis gene annotations. Thus, these sequences were obtained from the following sources: 25S rRNA (https://www.ncbi.nlm.nih.gov/nuccore/X52320.1) and the 5S rRNA database (http://combio.pl/rrna/). Contaminants were filtered out using STAR aligner (Dobin et al., 2013) with the following parameters: --outFilterMismatchNoverLmax 0.1 --outReadsUnmapped Fastx –outSAMmultNmax 1. After contaminant filtering, the remaining reads were mapped to the genome using STAR aligner using the following parameters: --outFilterMismatchNoverLmax 0.1 --alignIntronMax 8000 --outSAMmultNmax 1. Mapped reads were counted over annotated CDS using FeatureCounts (Liao et al., 2014) using the parameters: -M -s 1 -t CDS -g gene_id.
Tobacco: There are two available genomes for tobacco, which are both available from solgenomics (https://solgenomics.net/). The most recent version was used for alignment (Edwards et al., 2017) since it is conveniently assembled into chromosomes. Nuclear rRNA sequences used to filter the Ribo-seq libraries were obtained from NCBI; 26S (AF479172.1), 18S (AJ236016.1), 5.8S (AJ300215.1) and 5S (AJ222659.1). The tobacco chloroplast (NC_001879.2) and mitochondrial (NC_006581.1) genomes were also obtained from NCBI. Chloroplast and mitochondrial rRNA sequences were extracted from the respective genome annotations. The remainder of the analysis pipeline follows the same structure as what was performed for Arabidopsis.
Assembly: Arabidopsis: TAIR10 genome (https://plants.ensembl.org/index.html) using the Arabidopsis GTF gene annotations (v45)
Assembly: Tobacco: The Edwards genome assembly was used (solgenomics (https://solgenomics.net/). The chloroplast genome (NC_001879.2) and mitochondria genome (NC_006581.1) were obtained from NCBI.
Supplementary files format and content: "library_stats.xlsx" contains information on the proportion of reads mapping to rRNA, nuclear genome, plastid genome, and mitochondria genome.
Supplementary files format and content: "counts_over_CDS-Arabidopsis.xlsx" contains the number of reads mapping to annotated Arabidopsis coding sequences (CDS)
Supplementary files format and content: "counts_over_CDS-Tobacco.xlsx" contains the number of reads mapping to annotated Tobacco coding sequences (CDS)

Submission date

Mar 02, 2023

Last update date

Jul 05, 2024

Contact name

Reimo Zoschke

Organization name

Max Planck Institute of Molecular Plant Physiology

Lab

Zoschke Lab

Street address

Am Mühlenberg 1