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| Status |
Public on Mar 15, 2012 |
| Title |
Ribosome protected fragments Wild type 4hpf |
| Sample type |
SRA |
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| Source name |
Whole embryo, wild type
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| Organism |
Danio rerio |
| Characteristics |
sample type: ribosome protected fragments
tissue: whole embryo
genotype: wild type
developmental stage: 4hpf
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| Extracted molecule |
total RNA |
| Extraction protocol |
Ribosome protected fragments were obtained from 80 embryos per sample according to an adapted protocol from (Ignolia et al, 2009). Embryos were incubated with 0.1 ug/ml cycloheximide in Methylene blue water for 5 min, then snap frozen in 25ul Lysis buffer and clarified for 10 min at 10g 4degC. Supernatant was incubated with RNaseI and DNase at room temperature for 45 minutes with gentle mixing, then digested extracts (50ul) were overlain in a 100ul cushion of 1.8M sucrose in 20mM total Tris HCL ph8.0; 140mM KCl; 5 mM MgCl2; 100 ug/ml Cycloheximide; 0.5 mM DTT; 40U/ml SUPERas In (Ambion #AM2694) for 14 h at 55.000 rpm in a TLS55 rotor in a Beckman TL100 ultracentrifuge. The bottom 30 ul was mixed with 4ul of PNK buffer and 4 ul of Proteinase K and incubated at 37degC for 30 minutes. RNA was extracted using Trizol, then run on a 15% Urea gel to excise the 28-38 nt region. To construct barcoded Illumina small RNA libraries, samples were eluted overnight in 300 mM NaOAc pH 5.5; 1 mM EDTA; 0.1U/ul SUPERas In (Ambion #AM2694), followed by Ethanol precipitation. RFP and RNA input fragments were 3?-dephosphorylated with polynucleotide kinase in T4 polynucleotide kinase buffer (without ATP) for 1 h at 37degC, followed by 10 min incubation at 75degC and ethanol precipitation. Samples were resuspended and ligated to Illumina v1.5 3' adapter using T4 RNA Ligase 2, truncated K227Q, and RNAseOUT Recombinant Ribonuclease Inhibitor for 6h at 20degC, then separated on a 10% Urea gel to extract the 3' ligation products. These were 5' phosphorylated with polynucleotide kinase in T4 polynucleotide kinase buffer with 1mM ATP for 30 min at 37degC and precipitated in ethanol. 5' ligation was performed using T4 RNA Ligase 1 for 6 hrs at 20degC with individual 100uM barcoded 5' adapters, then separated on a 10% Urea gel. Gel-purified samples were reverse transcribed using SuperScript II Reverse Transcriptase according to the Invitrogen protocol, then amplified using Phusion High-Fidelity DNA Polymerase. PCR was carried out with an initial 30s denaturation at 98degC followed by 4 cycles of 30s denaturation at 98degC and 15s extension at 72degC, followed by 10-15 cycles of 10s denaturation at 98degC, 30s annealing at 50degC, and 15s extension at 72degC. Reactions were separated on a non-denaturing 8% polyacrylamide TBE gel and correct sized DNA fragments were sequenced using Illumina HiSeq2000. In parallel, RNA input from 20 embryos was extracted using Trizol, then poly(A) selected using biotinylated oligo(dT40) at room temperature and eluted. Samples were then alkaline fragmented for 20min at 95degC using 2 mM EDTA; 10 mM Na2CO3; 90 mM NaHCO3 pH 9.3; then neutralized with 560ul of ice-cold NaOAc (300mM) pH 5.5 and RNA precipitated with isopropanol. Small RNA sequencing libraries were constructed as above, selecting for fragments in the 30-50 nt range.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina Genome Analyzer II |
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| Data processing |
Ribo_Wt_4_A.bed; genome build: danRer7
Tophat v1.2.0 alignment to UCSC danRer7 (Zv9) genome sequence, guided by Ensembl r63 gene models; default settings, limited to reads mapping 5 or fewer times. Reads were trimmed of 3' adapter sequence
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| Submission date |
Dec 27, 2011 |
| 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 |
GPL9319 |
| Series (1) |
| GSE34743 |
Ribosome profiling of early zebrafish embryos -- miRNA-mediated regulation during embryogenesis causes translational repression before mRNA decay |
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| Relations |
| SRA |
SRX113358 |
| BioSample |
SAMN00769046 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM854440_Ribo_Wt_4_A.bed.gz |
2.0 Mb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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