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Sample GSM5203562

Query DataSets for GSM5203562

Status

Public on Sep 06, 2022

Title

Riboseq_E15.5_1

Sample type

SRA

Source name

neocortex

Organism

Mus musculus

Characteristics

treatment: RNAse-T1 & RNAse-A digestion
developmental stage: E15.5
tissue: neocortex

Growth protocol

Neocortex tissue was sub-dissected from embryos and pups from timed pregnant mice, as described in Kraushar et al. Molecular Cell. 81; 2021.

Extracted molecule

total RNA

Extraction protocol

Neocortex tissue was lysed on ice in 20 mM HEPES, 100 mM KCl, 7.5 mM MgCl2, pH 7.4, supplemented with 20 mM Dithiothreitol (DTT), 0.04 mM Spermine, 0.5 mM Spermidine, 1x Protease Inhibitor cOmplete EDTA-free (Roche, 05056489001), 0.3% v/v IGEPAL CA-630 detergent (Sigma, I8896) and clarified by centrifugation at 16100 xg for 5 min at 4 °C with a benchtop centrifuge. Samples were then measured for A260 ODU on a NanoDrop 1000 Spectrophotometer. For digestion of ribosome protected RNA fragments (RPFs), Ribo-seq samples were then mixed with 60U RNAse-T1 plus 96 ng RNAse-A per ODU, and incubated for 30 min at 25 °C, shaking at 400 rpm. To stop RNAse activity, 200 U of SUPERase-In RNAse inhibitor was then added.
Riboseq sequencing library preparation: the RNA was first ligated to a 3’ adapter 4N-RA3, and gel-purified using a 15 % denaturing urea-PAGE gel . Next the 5’ adapter OR5-4N was ligated and gel purified. The RNA was reverse transcribed and PCR-amplified by Phusion High-Fidelity DNA polymerase (Thermo Fischer, F-530XL). The cDNA was visualized on a 2.5 % agarose gel, a ~150 bp sized fragment was excised and purified by Zymoclean Gel DNA Recovery kit (Zymo Research, D4007/D4008).

Library strategy

OTHER

Library source

transcriptomic

Library selection

other

Instrument model

Illumina HiSeq 4000

Description

ribosome-protected mRNA fragment

Data processing

Library strategy: Ribo-Seq
Riboseq reads were stripped of adaptor sequences using cutdapt, and contaminants such as transfer RNAs (tRNAs) and rRNA were removed by alignment to a contaminants index via STAR 2.7.0, consisting of nucleotide sequences from known mouse rRNA and tRNA sequences drawn from the gencode annotation.
Unaligned reads from this analysis were then aligned to mouse GENCODE release M12 (mus musculus) with the STAR v 2.7.0 splice-aware alignment tool allowing for up to 1 mismatch. The star genome index was built using GENCODE M12. Only uniquely aligning reads were used.
The RiboseQC pipeline v1.1 (https://github.com/ohlerlab/RiboseQC) was used to confirm 3-nucleotide periodicity of the data and deduce P-site positions from the Riboseq reads.
Ribosome P-site count metaplots and fold change were calculated by first normalizing the P-site coverage track for each gene to the gene's total density, excluding genes with < 32 reads or less (low count filtering), and then all genes’ tracks were added together to generate a mean P-site density at each point for each sample. Confidence intervals were calculated by resampling from the 3 replicates for each condition.
Genome_build: GENCODE release M12 (Mus musculus)
Supplementary_files_format_and_content: tsv count table

Submission date

Mar 23, 2021

Last update date

Sep 06, 2022

Contact name

Matthew Lee Kraushar

E-mail(s)

matthew.kraushar@molgen.mpg.de

Organization name

Max Planck Institute for Molecular Genetics

Department

Department of Computational Molecular Biology