Illumina HiSeq 2500 paired end sequencing; Transcriptome profiling of mutant ASH2,RBBP5,WDR5 and WT(N2 or wild type) of C.elegans by RNASeq
Transcriptome profiling of mutant ASH2,RBBP5,WDR5 and WT(N2 or wild type) of C.elegans by RNASeq
We used four C. elegans strains: N2(wild type), wdr-5(ok1417) III, rbbp-5(tm3463) II, and ash-2(tm1905) II. Strains were kept at 20°C. 20 x 10cm plates of gravid adults were bleached and synchronised L1s were seeded on 2 X Corning tissue culture dishes (245 x 245 mm) seede with 20x OP50. When the synchronised young gravid mothers started to lay eggs, they were bleached to obtained embryos. These embryos were scored to ensure comparative stages between wild type and the mutants. About 10-12% of embryos are at <28-cell stage, 25-30% of embryos are between the 28-cell stage and 100-cell stage, 45-55% are between ~100-cell stage and pre-coma stage (~400 cell), and 5-15% are post-coma stage. Three independent biological replicates were prepared for RNA-seq Total RNA was extracted using TRIzol (Invitrogen) and samples were frozen at -80°C. First strand cDNA synthesis was performed using the SuperScript VILO cDNA Synthesis Kit (Invitrogen), according to the manufacturer's instructions. Illumina TruSeq Stranded mRNA Library Prep Kit