GSM4818854: Wild-type N2 replicate 3; Caenorhabditis elegans; RNA-Seq
For C. elegans total RNA isolation, nematodes collected from synchronized plates were washed three times with ice-cold S-Buffer and flash-frozen in liquid nitrogen. Afterwards, 500 µl ice-cold TRI Reagent (Molecular Research Center, #TR118) was added to the ground pellet. RNA was extracted based on the phenol-chloroform extraction method. Concentration of the resulting RNA was measured with an LVis plate on a CLARIOstar microplate reader (BMG LABTECH) and stored at -80 °C until further use. The quality of the isolated RNA was determined with a Qubit® (1.0) Fluorometer (Life Technologies) and a Bioanalyzer 2100 (Agilent). Only those samples with a 260 nm/280 nm ratio between 1.8–2.1 and a 28S/18S ratio within 1.5–2 were further processed. The TruSeq RNA Sample Prep Kit v2 (Illumina, #RS-122-2001/2) was used in the succeeding steps. Briefly, total RNA samples (100-1000 ng) were poly A enriched and then reverse-transcribed into double-stranded cDNA. The cDNA samples was fragmented, end-repaired and polyadenylated before ligation of TruSeq adapters containing the index for multiplexing Fragments containing TruSeq adapters on both ends were selectively enriched with PCR. The quality and quantity of the enriched libraries were validated using Qubit® (1.0) Fluorometer and the Caliper GX LabChip® GX (Caliper Life Sciences). The product is a smear with an average fragment size of approximately 260 bp. The libraries were normalized to 10 nM in Tris-Cl 10 mM, pH 8.5 with 0.1% Tween 20.