GSM1604425: FUDR-Treated Old (Day 12); Caenorhabditis elegans; RNA-Seq
Worms were lysed in QIAzol (QIAGEN) by beadbeating with 1.0 mm dia. zirconia/silica beads (BioSpec), four times, 1 min each time, with 2 minute pauses on ice. Total RNA was then extracted and purified with miRNeasy Mini Kit (QIAGEN). To generate libraries for RNA-seq, mRNA was purified from 5ug total RNA using the Dynabeads mRNA Direct kit (Ambion). We chose to fragment the mRNA to ~40-50bp rather than the conventional 200-300bp size using RNA fragmentation reagents from Ambion. To do this, the fragmented RNA was phosphorylated by T4 PNK (NEB) and then size selected on a 15% polyacrylamide gel containing urea. Following size selection, the entire RNA was used as input in the TruSeq small RNA sample preparation protocol (Illumina). Final size selection of amplified library was done using 6% polyacrylamide gels.
GEO Accession
GSM1604425