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Status |
Public on Apr 04, 2014 |
Title |
5h RPFs column replicate 2 run 1 |
Sample type |
SRA |
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Source name |
Whole embryos
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Organism |
Danio rerio |
Characteristics |
strain: TUAB Stage: 5hpf separation: column
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Growth protocol |
Embryos are incubated at 28C
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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.
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina HiSeq 2000 |
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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
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Submission date |
Dec 27, 2013 |
Last update date |
May 15, 2019 |
Contact name |
Antonio J. Giraldez |
E-mail(s) |
antonio.giraldez@yale.edu
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Phone |
203 785 5423
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Organization name |
Yale University
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Department |
Genetics
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Lab |
Giraldez Lab
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Street address |
333 Cedar Street
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City |
New Haven |
State/province |
CT |
ZIP/Postal code |
06510 |
Country |
USA |
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Platform ID |
GPL14875 |
Series (1) |
GSE53693 |
Identification of small ORFs in vertebrates using ribosome footprinting and evolutionary conservation |
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Relations |
BioSample |
SAMN02486856 |
SRA |
SRX399800 |
Supplementary data files not provided |
SRA Run Selector |
Raw data are available in SRA |
Processed data are available on Series record |
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