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| Status |
Public on May 23, 2013 |
| Title |
20120724_RPF-Seq_5dpf |
| Sample type |
SRA |
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| Source name |
Whole embryos
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| Organism |
Danio rerio |
| Characteristics |
developmental stage: 5 days post fertilization
strain: TL/AB
target molecule: ribosome footprinted RNA
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| Treatment protocol |
Embryos were quickly washed in ice cold PBS and flash frozen in liquid nitrogen
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| Growth protocol |
400-600 embryos per stage from TL/AB WT strains were allowed to grow at 28.5°C and staged according to Kimmel et al. Dev. Dyn. 1995
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| Extracted molecule |
total RNA |
| Extraction protocol |
Embryos were lysed by repeated micropipetting in 1.5ml of cold polysome buffer (20 mM Tris-HCl pH 7.4, 250 mM NaCl, 15 mM MgCl2, 1mM dithiothreitol, 100 μg/ml cycloheximide) with added 0.5% Triton X-100, 500 μg/ml GMP-PNP, 24 U/ml TurboDNase (Ambion AM2238), incubated with agitation for 10 min at 4°C, and clarified by centrifugation at 1300 rcf for 10 min at 4°C. 20μl RNAseI (Ambion AM2294) was added to the 1.5 ml of supernatant and incubated for 30 min at 37°C, then stopped by chilling on ice and addition of 40 μl of SuperaseIn (Ambion AM2694). Footprinted samples were pelleted through a sucrose cushion (1M sucrose in polysome buffer with added 100 U/ml SuperaseIn) by centrifugation at 260,000 rcf for 4.5 hours at 4°C, and resuspended in 800 μl 10mM Tris pH 7.4 with 1% SDS. RNA was purified by hot acid phenol/chloroform extraction and precipitated by standard ethanol precipitation.
Libraries were prepared essentially as described by Ingolia et al. Cell 2011
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
size fractionation |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
size-selected on PAGE gel, ~30nt
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| Data processing |
Adapters trimmed using custom script
Reads that mapped to zebrafish rRNAs (SILVA rRNA database: http://www.arb-silva.de/) by Bowtie2 -N 1 -L 20 _k 20) were discarded
Remaining reads mapped to previously assembled zebrafish developmental transcriptome (Pauli et al. 2012) on top of the Zv9/danRer7 assembly of the zebrafish genome by Tophat2; no indels, no novel junctions, -M -g 10
Only RPFs of length 27nt to 32nt used. P-site positions determined to be +12 for 27-28nt RPFs, +13 for 29-31nt RPFs, +13 for 29-31nt RPFs, +14 for 32nt RPFs.
Custom scripts used to generate bw files of genome-wide ribosome profiles corresponding to P-site occupancy, in conjunction with BedTools and UCSC bedgraphToBedBed
Genome_build: Zv9 (danRer7)
Supplementary_files_format_and_content: bigwig files corresponding to P-site occupancy by ribosomes
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| Submission date |
Apr 30, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Guo-Liang Chew |
| E-mail(s) |
chew@fas.harvard.edu
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| Organization name |
Harvard University
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| Department |
Dept of Molecular and Cellular Biology
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| Lab |
Schier
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| Street address |
16 Divinity Ave BL 1019
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| City |
Cambridge |
| State/province |
MA |
| ZIP/Postal code |
02139 |
| Country |
USA |
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| Platform ID |
GPL14875 |
| Series (1) |
| GSE46512 |
Ribosome Profiling over a Zebrafish Developmental Timecourse |
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| Relations |
| BioSample |
SAMN02087564 |
| SRA |
SRX272888 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1131537_20120724_RPF-Seq_5dpf_fwd.bw |
8.0 Mb |
(ftp)(http) |
BW |
| GSM1131537_20120724_RPF-Seq_5dpf_rev.bw |
10.2 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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