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| Status |
Public on Nov 04, 2011 |
| Title |
L3_STAGE_GVL4R4202 |
| Sample type |
SRA |
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| Source name |
L3
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| Organism |
Caenorhabditis elegans |
| Characteristics |
strain: N2
developmental stage: L3
purification method: myo-3::PABP pulldown
tissue: muscle
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| Treatment protocol |
To crosslink poly(A) RNA with FLAG-PABP, worms were treated with 1% for-maldehyde in M9 for 15 min at 20°C . The formaldehyde was then inactivated by washing with 125 mM glycine for 5 min at 20°C and then with TBS (4X). The worms were then stored frozen until library preparation.
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| Growth protocol |
myo-3-FLAG-PABP-SL2-GFP::unc-54 3’UTR transgenics worms were grown on 15cm2NGM (Nematode Growth Media) plates seeded with HB101 and synchronized by alkaline/hypochride. Starved L1 larvae were then plated on 15 cm2 plates (HB101 as food) raised at 20°C. Samples were taken at ~20hr (L2), ~30hr (L3), ~45hr (L4), ~72hr (gravids). Worms were collected in M9, washed 1X with M9 for 15 min to remove residue bacteria and then with milliQ water for 5 min.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Worms were lysed in lysis buffer (50mM HEPES-KOH pH7.4, 1mM EDTA, 140mM KCL, 10% Glycerol, 0.5% NP-40, 1mM DTT, Protease inhibitor cocktail tablet, 1000U/ml of ribonuclease inhibitor and 20mM RVS, Sigma). Worm debris were removed by spinning at 18,000g for 20 min. The clear supernatant was then transferred to new tube and protein concentration was adjusted to 10mg/ml and incubated with 100ul of anti-FLAG M2 beads (Sigma) for 2 hrs at 4°C on nutator. After 2hrs of incubation the lysates washed with lysis buffer (3X), then with wash buffer (2X) and finally with TE buffer (1X). Elution was done using 100ul of elution buffer (2X) by incubation at 65°C for 5 min in a thermo-mixer. The eluted complex was then incubated for 6hrs at 65°C to reverse formaldehyde crosslinking. Proteins were digested using 10mg/ml of proteinase K by incubating at 37°C for 30min. Nucleic acid was extracted using TRI-Reagent (Applied Biosystems) followed by isopropanol extraction and ethanol precipitation. 3’UTR libraries were made using the protocol published in The Landscape of C. elegans 3′UTRs . Mangone, et al., 329 (5990): 432-435.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
454 GS FLX Titanium |
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| Data processing |
GRAVID_STAGE_GVOZ88J02.fna: is an intermediate, tab-delimited file derived from the GRAVID_STAGE_GVOZ88J02.sff raw sequence read file with the format: 454 Read ID, full read sequence, and linker-removed sequence. Linker sequence was removed using a custom Perl script.
mya-3__PABP_L2_STAGE_GVL4R4201.fna: is an intermediate, tab-delimited file derived from the mya-3__PABP_L2_STAGE_GVL4R4201.sff raw sequence read file with the format: 454 Read ID, full read sequence, and linker-removed sequence. Linker sequence was removed using a custom Perl script.
L3_STAGE_GVL4R4202.fna: is an intermediate, tab-delimited file derived from the L3_STAGE_GVL4R4202.sff raw sequence read file with the format: 454 Read ID, full read sequence, and linker-removed sequence. Linker sequence was removed using a custom Perl script.
L4_STAGE_GVOZ88J01.fna: is an intermediate, tab-delimited file derived from the L4_STAGE_GVOZ88J01.sff raw sequence read file with the format: 454 Read ID, full read sequence, and linker-removed sequence. Linker sequence was removed using a custom Perl script.
fasta_PABP_Run2-seq.txt: is a consolidation of all four sequence libraries into a single, uniqued (based on sequence) FASTA-formatted sequence file, suitable for input to BLAT for alignment to the C. elegans genome (version WS190). Sequence reads less than 15 nt in length, and those reads with no discernable linker sequence were discarded prior to consolidation of the four sequence libraries. Identical reads (based on sequence identity) were collapsed into a single, unique read. All reads were assigned a unique identifier.
fasta_PABP_Run2-seq.txt.2bit.psl: The consolidated read file fasta_PABP_Run2-seq.txt was aligned to the WS190 C. elegans genome using the BLAST-Like Alignment Tool (description here: http://genome.ucsc.edu/FAQ/FAQblat.html) with a maximum intron of 1000, minimum window size of 5, and maximum gap of 6. Best matches were selected, and multiple alignments reported if present in more than one genomic location.
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| Submission date |
Nov 03, 2011 |
| Last update date |
May 15, 2019 |
| Contact name |
Ryan Mills |
| E-mail(s) |
remills@med.umich.edu
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| Phone |
734-647-9628
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| Organization name |
University of Michigan
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| Department |
Computational Medicine and Bioinformatics
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| Lab |
Ryan E. Mills, Ph.D.
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| Street address |
100 Washtenaw Ave
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| City |
Ann Arbor |
| State/province |
MI |
| ZIP/Postal code |
48109 |
| Country |
USA |
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| Platform ID |
GPL14832 |
| Series (1) |
| GSE33431 |
Comprehensive assembly and spatio-temporal analysis of the C. elegans 3' UTRome through isolation and sequencing of 3'UTRs in germline-dereived cell types and somatic tissues. |
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| Relations |
| SRA |
SRX104175 |
| BioSample |
SAMN00749948 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM826899_tab_L3_STAGE_454Reads.fna.gz |
10.5 Mb |
(ftp)(http) |
FNA |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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