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Purification of TAP::ALG-1 complexes and associated RNA from mixed-stage TAP::ALG-1 transgenic (WS4303) and wild-type (N2) animals

ABSTRACT: To discover new miRNA targets, we generated a C. elegans transgenic line expressing a functional N-terminally Tandem Affinity Purification (TAP) tagged ALG-1 protein We purified TAP::ALG-1 complexes from mixed-stage TAP::ALG-1 transgenic (WS4303) and wild-type (N2, serving as a mock control) animals and hybridized the associated mRNAs to two-color microarrays

ORGANISM(S): Caenorhabditis elegans

PROVIDER: GSE32941 | GEO | 2012/03/01

SECONDARY ACCESSION(S): PRJNA154397

REPOSITORIES: GEO

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Purification of TAP::ALG-1 complexes and associated RNA from mixed-stage TAP::ALG-1 transgenic (WS4303) and wild-type (N2) animals

Project description:To discover new miRNA targets, we generated a C. elegans transgenic line expressing a functional N-terminally Tandem Affinity Purification (TAP) tagged ALG-1 protein We purified TAP::ALG-1 complexes from mixed-stage TAP::ALG-1 transgenic (WS4303) and wild-type (N2, serving as a mock control) animals and hybridized the associated mRNAs to two-color microarrays RNA isolated from WS4303 and N2 (mock control) animals was analyzed for each IP sample and for each total worm extract sample. Three independent biological replicates have been performed. Long-oligo whole-genome C. elegans arrays, produced by the Genome Sequencing Center at Washington University in St. Louis (http://genome.wustl.edu/genome/celegans/microarray/ma_gen_info.cgi), were used for analysis. Log2 ratios (immunopurified RNA over total RNA from extract) were calculated for each gene. Microarrays generated from WS4303 animals were compared to microarrays generated from N2 mock control animals by a Student’s two sample t test.

2012-03-01 | E-GEOD-32941 | biostudies-arrayexpress

Microarray analysis of TAP::ALG-1 associated RNAs isolated from synchronized 'wild-type' animals and 'mir-58' mutants

2012-03-01 | GSE32942 | GEO

Microarray analysis of TAP::ALG-1 associated RNAs isolated from synchronized 'wild-type' animals and 'mir-58' mutants

Project description:To discover new miRNA targets, we generated a C. elegans transgenic line expressing a functional N-terminally Tandem Affinity Purification (TAP) tagged ALG-1 protein (C. elegans strain WS4303). We crossed the TAP::ALG-1 transgene into the mir-58(n4640) mutant background to generate the strain WS5041. For simplicity, we will hereafter term the TAP::ALG-1 transgenic animals as wild typeand the transgenic WS5041 animals as mir-58. We compared the mRNA population that coimmunopurified with TAP::ALG-1 from synchronized L4 stage wild-type animals with that from synchronized L4 stage mir-58 mutant animals by one-color Affymetrix gene arrays. miR-58 target mRNAs should be specifically underrepresented in the latter samples. Strains WS4303 (wt) and WS5041 (mir-58) were used for TAP::ALG-1 IPs. All experiments were conducted in three independent replicates. For each replicate, WS4303 and WS5041 were grown in parallel. 150 ng of TAP::ALG-1 associated RNA isolated from synchronized late L4 animals were sent to the GeneCore facilty in Heidelberg, Germany (http://www.genecore.embl.de/index.cfm), and the microarray data were generated according to their standard protocol (Weinmann et al. 2009).

2012-03-01 | E-GEOD-32942 | biostudies-arrayexpress

RNA-seq on D5 C. elegans in WT(N2), alg-1(gk214), alg-2(ok304)

Project description:In order to examine the misregulation of genes in loss of function alg-1 and alg-2 versus WT, RNA-seq was performed on D5 C.elegans in WT(N2), alg-1(gk214), alg-2(ok304)

2018-05-01 | GSE112753 | GEO

Expression analysis of WS4303 (wild type) vs WS5041 (mir-58 mutants) total mRNA

2012-03-01 | GSE32943 | GEO

ALG-1 and ALG-2

Project description:ALG-1 and ALG-2

| PRJNA448930 | ENA

Gene expression profile of alg-3(tm1155);alg-4(ok1041) animals compared to wild type

Project description:Gametogenesis is thermosensitive in numerous metazoa ranging from worms to man. In C. elegans a variety of germ-line nuage- (P-granule) -associated RNA-binding proteins including the Piwi-clade Argonaute, PRG-1, have been implicated in temperature-dependent fertility. Here, we describe the role of two AGO-class paralogs, alg-3 (T22B3.2) and alg-4 (ZK757.3) in promoting male fertility at elevated temperatures. A rescuing GFP::alg-3 transgene is localized in P-granules beginning at the late pachytene stage of male gametogenesis. alg-3/4 double mutants lack a subgroup of small RNAs, named 26G-RNAs, which target and appear to down-regulate numerous spermatogenesis-expressed mRNAs. These findings add to a growing number of AGO pathways required for temperature-dependent fertility in C. elegans and support a model in which AGOs and their small RNA co-factors function to promote robustness in gene-expression networks.

2010-02-24 | GSE18719 | GEO

Expression analysis of WS4303 (wild type) vs WS5041 (mir-58 mutants) total mRNA

Project description:To discover new miRNA targets, we generated a C. elegans transgenic line expressing a functional N-terminally Tandem Affinity Purification (TAP) tagged ALG-1 protein (C. elegans strain WS4303). We crossed the TAP::ALG-1 transgene into the mir-58(n4640) mutant background to generate the strain WS5041. For simplicity, we will hereafter term the TAP::ALG-1 transgenic animals as “wild type” and the transgenic WS5041 animals as “mir-58”. In addition to immunopurifying the TAP::ALG-1 and associated RNAs from these strains, we also compared total mRNA levels between these two strains. Total RNA was isolated from the same WS4303 and WS5041 total extracts that was further used for the TAP::ALG-1 RIP. Three independent biological replicates were analyzed. Long-oligo whole-genome C. elegans arrays, produced by the Genome Sequencing Center at Washington University in St. Louis (http://genome.wustl.edu/genome/celegans/microarray/ma_gen_info.cgi), were used for these experiments. A total of 10 µg of total RNA was used for cDNA synthesis.

2012-03-01 | E-GEOD-32943 | biostudies-arrayexpress

Developmental characterization of the microRNAs-specific C. elegans Argonautes alg-1 and alg-2

Project description:This study analyzes miRNA association with ALG-1 and ALG-2 in different stages during larval development of C. elegans Staged animals (L1, L2, L3, L4) - the alg-2 mutant expressing tagged-ALG-2, alg-1 mutant, and wild type controls - were lysed. ALG-1 IP or ALG-2 (GFP) IP was performed using tags from all four samples at all four timepoints. Small RNAs were released from antibodies and 5' end-labelled with AlexaFluor532. Labelled RNA was hybridized to a custom microRNA microarray platform to quantify miRNA content.

2012-02-03 | E-GEOD-35505 | biostudies-arrayexpress

miRNAs associated with ALG-1 and ALG-2 in adult C. elegans

Project description:Investigated the specific miRNAs that are assocaited with ALG-1 and ALG-2 in adult (D5) C. elegans. ALG-1 and ALG-2 were IP-ed from D5 collected C.elegans, RNA was isolated and used for small RNA sequencing. Two biological replicates were used.

2018-05-01 | GSE112754 | GEO

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