Assay summary for RNA-seq_Busch-Nentwich_Lab_0003AS
Busch-Nentwich Lab, Wellcome Trust Sanger Institute
Whole embryos collected in deep well 2ml round well blocks were snap frozen on dry ice and stored at Ð80. They were mechanically lysed in 100 microlitres RLT buffer containing 1 microlitre of 14.3M beta mercaptoethanol (Sigma) using a 5mm steel bead on a Qiagen TissueLyser for 5 mins at 10Hz. The lysate was allowed to bind to 1.8 volumes of Agencourt RNAClean XP (Beckman Coulter) beads for 10 mins. The plate was then applied to a plate magnet (Invitrogen) until the solution cleared and the supernatant was removed without disturbing the beads. While still on the magnet the beads were washed thrice with 70% ethanol and nucleic acid was eluted from the beads as per the manufacturerÕs instructions. Samples from each row on the plate were pooled and DNase treated for 20 mins at 37 Celsius followed by addition of 1 microlitre 0.5M EDTA and inactivation at 75 Celsius for 10 mins to remove residual DNA. RNA was then cleaned using 2 volumes of Agencourt RNAClean XP (Beckman Coulter) beads under the standard protocol.