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Sample GSM1299085

Query DataSets for GSM1299085

Status

Public on Apr 04, 2014

Title

5h RPFs column replicate 1 run 2

Sample type

SRA

Source name

Whole embryos

Organism

Danio rerio

Characteristics

strain: TUAB
Stage: 5hpf
separation: column

Growth protocol

Embryos are incubated at 28C

Extracted molecule

total RNA

Extraction protocol

For ribosome profiling, 50 wild type embryos for each condition were collected at a given stage. Embryos were lysed using 800ul of a mammalian cell lysis buffer containing 100ug/ml Cycloheximide as per the manufacturer’s instruction (ARTseq Ribosome Profiling Kit, RPHMR12126, Epicentre). For nuclease treatment, 3ul of ARTseq Nuclease was used. Ribosome protected fragments were run and 28-29nt fragments were gel purified as previously described in (Bazzini et al., 2012) and cloned according to the manufacturers protocol (ARTseq Ribosome Profiling Kit, RPHMR12126, Epicentre). For RNA input, total RNA was isolated from 400 ul of the 800 ul of clarified extract before ART- seq Nuclease treatment. Poly-A selection was done according to manufacturer guide- lines (Dynabeads mRNA Purification Kit, Cat no.610.06), and RNA fragmented using the Artseq Ribosome Profiling Kit Mammalian protocol.
Both RPF and RNA input fragments were cloned according to the Artseq Ribosome Profiling Kit, Mammalian. The final PCR was carried out with an initial 15 second denaturation at 98 °C, followed by 9-12 cycles of 15 seconds at 98 °C, 5 seconds at 55 °C and extension at 72 °C for 10 s. Reactions were separated on a non-denaturing 8% polyacrylamide TBE gel and DNA fragments of the correct size were extracted and sequenced.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Data processing

Base calling was performed using CASAVA-1.8.2.
The Illumina TruSeq adaptor sequence was then trimmed from raw reads by aligning its sequence, requiring 100% match of the first five base-pairs and a minimum alignment score of 60 (Matches:5, Mismatches:-4, Gap opening:-7, Gap extension:-7). Trimmed reads were then depleted of rRNA, tRNA, snRNA, snoRNA, and misc_RNA from Ensembl and RepeatMasker annotations using strand-specific alignment performed with Bowtie2 v2.1.0 with default parameters.
Filtered reads were aligned strand-specific to the Zebrafish Zv9 genome assembly using Tophat v2.0.8 with default parameters and the exon-junction coordinates from Ensembl r70.
Using the spliced version of each transcript (ensembl r73, Pauli et al lincRNAs, Ulitsky et al lincRNAs) we defined all ORFs as stop codon to most distal in-frame AUG codon without an intervening stop. Read coverage and counts were calculated using 28 & 29nt reads. For position-related calculation of ORFScore and Frame1 coverage, we counted reads only with the +12 position within the interior of the ORF (defined as the region excluding the reads aligning to the start and stop(-1) codons). RPKM values were calculated using input mRNA per transcript, normalizing WITHIN but not between transcript sets (total reads mapping to ensembl r73 or either lincRNA set). ORFScore and coverage values were calculated as outlined in Bazzini et al, 2013. Sequencing data were combined for each timepoint.
Genome_build: Zv9/danRer7
Supplementary_files_format_and_content: allinfo_final_ORFs.txt :: orfID=unique ID of ORF; geneID; transcriptID; startLoc & stopLoc=1-based transcript coordinates of ORF; txCDSStart&txCDSEnd=start and end of annotated CDS; Source=transcript source; orfFrame=ORFScore; orfRate=# of reads in ORF; covF1Prop=proportion of in-frame positions with aligning read(s); tx_Input_RPKM=RPKM of transcript from input mRNA

Submission date

Dec 27, 2013

Last update date

May 15, 2019

Contact name

Antonio J. Giraldez

E-mail(s)

antonio.giraldez@yale.edu

Phone

203 785 5423

Organization name

Yale University