Illumina HiSeq 2000 sequencing; RNA-Seq of Caenorhabditis elegans Mi2-depleted embryos at 24- and 100-cell stages
RNA-Seq of Caenorhabditis elegans Mi2-depleted embryos at 24- and 100-cell stages
wild-type or chd-3(eh4) L4 larvae were shifted to controlGFP or let-418(RNAi) plates and incubated 24h at 25°C. Gravid hermaphrodites were dissected and collected embryos were incubated at 25°C to pursue development up to the 24-cell or the 100-cell stage. In total 3 biological replicates from 3 different RNAi batches were prepared for each sample.  Total RNA was extracted with guanidium thiocyanate-phenol-chloroform (Qiazol®) using MaXtract High Density tubes (Qiagen) to ease phase separation and linear polyacrylamide (GeneElute LPA, 25mg/ml solution, Sigma) was used as RNA carrier at 5mg/300ml sample. RNA was precipitated with isopropanol and dissolved in RNase-free H2O. The samples were treated with DNase I (Qiagen RNase-Free DNase Set) and purified with the RNeasy MinElute Cleanup Kit (Qiagen). Pure RNA was finally dissolved in H2O. RNA quality and quantity were analyzed with an Agilent 2200 TapeStation using the High Sensitivity R6K kit. The 24 RNA samples were sent to the Lausanne Genomic Technologies Facility. cDNA libraries were prepared using 2.5ng of RNA following the Illumina TruSeq DNA Sample Prep protocol
Experimental Factor: chd-3 mutant
genotype
Experimental Factor: 100-cell embryo
developmental stage