title: WT_46_47
organism: Mus musculus
description: Protocols: Two male mice of matched genotype were used per purification. The animals, aged between 8-10 weeks. were sacrificed, testes extracted and albuginea removed. The seminiferous tubules were digested for 15 minutes at 35C with 1mg/ml of collagenase in PBS, supplemented with lactate and glucose. The cells were washed and further dissociation was performed by vigorous pipetting for 10 minutes on ice. The cells were twice filtered thought a 100µm filter and loaded onto the 2-4% BSA gradient (in 360ml). The sedimentation of cells was allowed to occur for 75 minutes. The cells were collected into 10-ml fractions and each fraction was analysed under the phase contrast microscope. The fractions that contained only round spermatids were pooled together and used for mRNA purification. Three biological replicates for each genotype were obtained by merging cells from two individuals. 4x106 cells for each replica were used for RNA purification by Trizol. Prior to purification, ERCC RNA spike-in Mix 1 (diluted 1:10) was added to each sample that were already lysed in Trizol. Purified RNA was resuspended in 50µl of sterile MQ water. 15µl of each sample were subjected to Turbo DNase treatment. 3µl of DNase treated RNA was used for TruSeq Stranded mRNA LT Illumina library preparation following manufacturer's protocol