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| Status |
Public on Mar 01, 2012 |
| Title |
WS5041 (mir 58(n4640); alg 1(tm492); opIs205). Replicate 3 |
| Sample type |
RNA |
| |
|
| Source name |
mir-58(n4640) mutant expressing TAP::ALG-1
|
| Organism |
Caenorhabditis elegans |
| Characteristics |
strain: WS5041
genotype: mir-58(n4640); alg-1(tm492); opIs205
transgene: opIs205[Peft 3::TAPtag::alg 1(genomics+3’UTR); unc 119(+)] integrated by bombardment
developmental stage: synchronized, late L4
tissue: whole animal
|
| Treatment protocol |
The coding sequence for the N-terminal tandem affinity purification (TAP)-tag was amplified from plasmid pBS1761 (Rigaut et al. 1999) by PCR. The alg-1 genomic sequence was amplified from genomic C. elegans DNA by PCR. The sequences were tagged with the following restriction sites: 5’AscI:TAP-tag:3’FseI; 5’FseI:alg-1(genomic+3’UTR):3’PacI. The tagged fragments were subcloned into the pCR2.1 TOPO vector (Invitrogen). Both constructs were then inserted into pLN022 (Neukomm et al. 2011) to generate the final expression vector pMJ001, where TAP::ALG-1 expression is under the control of the ubiquitous eft-3 promoter. Low copy transgenic animals were generated by bombardment as previously described (Praitis et al. 2001). The protocol was adapted as follows: unc-119(ed3) mutant worms were grown in liquid culture. pMJ001 was precipitated onto gold beads and shot at unc-119(ed3) mutant worms using a Biolistic PDS-1000 bombardment apparatus (Bio-Rad). Bombarded worms were distributed onto large seeded plates and left to starve. Starved worms were chunked onto fresh seeded plates and screened for unc–119(+) transgenic animals. Rescued worms that expressed TAP::ALG-1 were kept and analyzed. All further experiments were done with the integrant opIs205[Peft-3::TAPtag::alg-1(genomics+3’UTR); unc-119(+)]. The transgenic line carrying opIs205 was crossed into alg-1(tm492) mutant animals to generate the strain WS4303 (alg-1(tm492); opIs205). The opIs205 transgene successfully rescued the reduced brood size of homozygous alg-1(tm492) mutant animals, proving the functionality of our transgene (data not shown).WS4303 was also crossed into mir-58(n4640) animals to generate the strain WS5041 (mir-58(n4640); alg-1(tm492); opIs205).
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| Growth protocol |
For synchronized late L4 stage cultures of transgenic animals, eggs were isolated by bleaching (Stiernagle 2006). The purified eggs were allowed to hatch overnight on large unseeded NGM plates. Synchronized L1 larvae were then spotted onto large NGM plates seeded with OP50 and grown at 25°C until late L4 stage (36 hours).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA Immunopurifications (RIPs) have been described previously (Gerber et al. 2004). We have adapted the protocol accordingly. In brief, after harvesting, worms were separated from the bacteria by several washes in ice-cold buffer A (20 mM Tris–HCl [pH 8.0], 140 mM KCl, 1.8 mM MgCl2, 0.1% Nonidet P-40 (NP-40), 0.1 mg/ml heparin) and frozen in liquid nitrogen and stored at -80°C until further use. 1 ml of frozen worm pellet was resuspended in 5 ml buffer B (buffer A plus 1.5 mM dithiothreitol (DTT), 1 mM phenylmethylsulfonylfluoride, 0.5 µg/ml leupeptin, 0.8 µg/ml pepstatin, 20 U/ml DNase I, 100 U/ml RNaseOUT (Invitrogen), and 0.2 mg/ml heparin). The resuspended worms were drop wise refrozen in liquid nitrogen and homogenized by a TissueLyser instrument (Qiagen) by 4 cycles of 4 minutes each with a setting of 30 Hertz; the metal chars (50 ml) containing the samples were always refrozen in liquid nitrogen between the cycles. The worm lysates were clarified by sequential centrifugation steps and the purified extracts were incubated on a rotor with 400-µl slurry (50% (v/v)) IgG–agarose beads (Sigma) for 2 h at 4°C. The beads were washed four times for 15 min at 4°C with buffer C (20 mM Tris–HCl (pH 8.0), 140 mM KCl, 1.8 mM MgCl2, 1 mM DTT, 0.01% NP-40, 10 U/ml RNasin). ALG-1 was released from the beads by incubation with 100 U of TEV protease (Invitrogen) for 90 min at 25°C. RNA was isolated from the TEV eluates (IP fraction) by extraction with the miRVana kit (Ambion) according to the vendor’s instructions. RNA was quantified with a Nanodrop device (Witeg AG).
|
| Label |
biotin
|
| Label protocol |
100 ng of RNA were amplified by the MessageAmp™ aRNA Kit (Ambion) in a two cycle protocol and labeled as recommended by the vendor.
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| |
|
| Hybridization protocol |
According to Affymetrix protocol
|
| Scan protocol |
Arrays were scanned on an Affymetrix 3000 7G scanner, according to manufacturer instructions.
|
| Description |
TAP::ALG-1 associated RNA (RIP)
|
| Data processing |
Microarray data were preprocessed using the rma algorithm of the Bioconductor package affy (Gautier et al. 2004). 15880 identifiers out of the total of 22625 identifiers passed the log2 signal threshold of 4.644 (=linear signal threshold of 25) and were further considered. To identify transcripts that were specifically enriched by association to TAP::ALG 1 isolated from WS4303 animals compared to WS5041 animals, we performed a two class paired Significance Analysis of Microarrays (SAM) (Tusher et al. 2001).
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| |
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| Submission date |
Oct 12, 2011 |
| Last update date |
Mar 01, 2012 |
| Contact name |
Marko Jovanovic |
| E-mail(s) |
markojov@broadinstitute.org
|
| Organization name |
Broad Institute of MIT and Harvard
|
| Department |
Core Group (Regev group) member
|
| Lab |
Regev lab
|
| Street address |
7 Cambridge Center
|
| City |
Cambridge |
| State/province |
MA |
| ZIP/Postal code |
02142 |
| Country |
USA |
| |
|
| Platform ID |
GPL200 |
| Series (2) |
| GSE32942 |
Microarray analysis of TAP::ALG-1 associated RNAs isolated from synchronized 'wild-type' animals and 'mir-58' mutants |
| GSE32944 |
Identification of miRNA target genes in C. elegans by RIP-chip-SRM |
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