Caenorhabditis elegans
Protocols: The following C. elegans strains were obtained from the Caenorhabditis Genetic Center, funded by the NIH Office of Research Infrastructure Programs (P40 OD010440): N2 Bristol wild type, DA1116 (eat-2(ad1116)II), MQ887(isp-1(qm150)IV), RB754 (aak-2(ok524)X), VC222 (raga-1(ok386)II), and RB1206 (rsks-1(ok1255)III), (three latter strains made by groups part of the International C. elegans Knockout Consortium). The generation of the CA AMPK strain was previously described 22,38 . CF1038 (daf-16(mu86)I) and CF1041 (daf-2(e1370)III) were obtained from the Kenyon lab via the Dillin lab. The SS104 (glp-4(bn2)) strain was a gift from K. Blackwell lab. The splicing reporter strain KH2235 (lin-15(n765)ybIs2167[eft-3::ret-1E4E5(+1)E6-GGS6-mCherry+eft-3::ret-1E4E5(+1)E6(+2) GGS6-EGFP+lin-15(+)+pRG5271Neo]X) was a gift from Hidehito Kuroyanagi. The inverted splicing reporter WBM535 was made by injecting modified minigene reporter plasmids into N2 worms. The plasmids were made by deleting the (+2) frameshift in the EGFP minigene and inserting a (+2) frameshift into the mCherry minigene. Worms were grown and maintained on standard nematode growth media (NGM) seeded with E. coli (OP50-1). E. coli bacteria were cultured overnight in LB at 37°C, after which 100 μl of liquid culture was seeded on plates to grow for 2 days at room temperature. All RNAi constructs came from the Ahringer RNAi library, except hrp-2 and hrpf-1 RNAi constructs, which originated from the Vidal RNAi library. RNAi experiments were carried out using E.coli HT115 bacteria on standard NGM plates containing 100μg/ml Carbenicillin. HT115 bacteria expressing RNAi constructs were grown overnight in LB supplemented with 100μg/ml Carbenicillin and 12.5μg/ml Tetracycline. NGM plus Carbenicillin plates were seeded 48 hours before use. Respective dsRNA expressing HT115 bacteria were induced by adding 100μl IPTG (100mM) one hour before introducing worms to the plate. RNAi was induced for all experiments from egg hatch. Total RNA was extracted using Qiazol reagent (QIAGEN), column purified by RNeasy mini kit (QIAGEN) according to manufacturer's instructions. cDNA was synthesized using Quantitect Reverse Transcriptase kit (QIAGEN). cDNA libraries were prepared from 1μg total RNA using TruSeq RNA Sample preparation v2 kit (Illumina).
ENA first public
2016-11-29
ENA last update
2016-06-27
External Id
SAMEA4051784
INSDC center alias
Department of Biochemistry and Molecular Biology University of Southern Denmark
INSDC center name
Department of Biochemistry and Molecular Biology University of Southern Denmark
INSDC first public
2016-11-29T17:01:13Z
INSDC last update
2016-06-27T17:40:18Z
INSDC status
public
Submitter Id
E-MTAB-4866:day3_N2_sfa1_repl2
age
3
broker name
ArrayExpress
rna interference
Sfa-1
sample name
E-MTAB-4866:day3_N2_sfa1_repl2
strain
N2