strain: daf-2(e1379) mutant developmental stage: Adult age: Day 10
Growth protocol
Synchronized L1s of N2 (wild-type) and mutant daf-2(e1370) strains were fed on OP50 and grown at 16oC until L4 stage to avoid dauer formation in the daf-2 temperature sensitive mutants. 5-fluorodeoxyuridine (FUDR) (0.1g/ml) was added to the plates to prevent progeny production and the plates where shifted to 25oC. FUDR does not affect lifespan of C. elegans adults [6, 7] (and Supplementary Figure 4). Day 0 worms were harvested ~20 hours after addition of FUDR and 25oC temperature shift. The remaining worms were kept at 25oC for 4 days, and then moved to 20oC. Day 10 worms were harvested 10 days after the addition of FUDR
Extracted molecule
total RNA
Extraction protocol
18-26-nucleotide small RNAs from 85 - 305 micrograms of TOTAL RNA were gel extracted from denaturing 15% PAGE. Bands were visualized by the addition of trace amounts of radiolabelled 24-nt and 18-nt RNA markers. After elution and precipitation, a pre-adenylated-3'adaptor (miRNA cloning linker from IDT) was ligated (T4 RNA ligase) to the 5'-phosphate of small RNAs, purified by PAGE and bands representing ~35-41-nt bands were excised, eluted and precipitated. A 5'-adaptor was then ligated, followed by PAGE purification, and bands representing ~52-58-nt bands were excised, eluted and precipitated. 1st strand cDNA synthesis of the samples was done with Superscript III and primer specific to adaptor sequence. cDNA was PCR amplified, digested with BanI, and finally concatemerized with T4 DNA ligase (30 minutes at room temperature). The concatemerized samples were purified with Min. Elute PCR Purification Kit (Qiagen), thus removing salts, enzyme, primers and dsDNA <70bp while eluting the desired dsDNA in size range >70bp. The quality of the dsDNA was confirmed on nanospec (A260/280 > 1.8) and by running 300ng on 2% Nusieve GTG Gel, showing a smear of concatemers from ~70bp-600bp. Purified concatemers were then submitted for deep sequencing to 454 Lifesciences/Roche.
Library strategy
RNA-Seq
Library source
transcriptomic
Library selection
size fractionation
Instrument model
454 GS
Description
C.elegans genome WS190
Data processing
Sequences corresponding to cloning and sequencing adapters or size-selection markers were trimmed from raw data. Sequencing reads were then aligned to the C. elegans genome (version WS190), using NCBI megablast (BLAST version 2.2.17 with the following options: -W 12 -p 100. Only perfect alignments were retained (full length, 100% identity). The number of sequence reads that correspond to known miRNAs was assessed using perfect sequence matching to a database of known miRNAs (miRBase, v.13.0). To compare the differential expression of miRNAs across samples, the number of miRNA reads in each sample was normalized to the total number of reads in each sample that matched the C. elegans genome.
