title: GSM5099806: ADMSO_2; Mus musculus; RNA-Seq
library_construction_protocol: Cells were lysed, homogenized and collected using 1 ml of RNAzol®RT before adding 0.4 ml of water per 1 ml of homogenate. The mixture was vortexed for 5 min and centrifuged at 12,000 g for 15 min. Following centrifugation, DNA, proteins and polysaccharides form a semisolid pellet at the bottom of the tube. The RNA remains soluble in the supernatant. The supernatant was transferred to a new tube and 1 volume of 70% ethanol was added. The mixture was homogenized by pipetting. Samples were then applied to a RNeasy spin column and RNAs were purified according to Qiagen's instructions. Base conversion was performed using the Quant-seq 3' mRNA-Seq library preparation kit. mRNA-Seq libraries were generated according to manufacturer's instructions from 500 ng of total RNA using the QuantSeq 3′mRNA‐Seq Library Prep Kit for Illumina (FWD) (# 015, Lexogen GmbH, Vienna, Austria). Reverse transcription was initiated by oligo dT priming. After first strand cDNA synthesis the RNA was removed and second strand synthesis was initiated by random priming. Oligo dT primer and random primers contain Illumina-compatible adapter sequences. The resulting double-stranded cDNA was then purified and PCR amplified (30 sec at 98oC; [10 sec at 98oC, 20 sec at 65oC, 30 sec at 72oC] x 11 cycles; 1 min at 72oC), introducing i7 indexes. Surplus PCR primers were further removed by purification using SPRI-select beads (Beckman-Coulter, Villepinte, France) and the final libraries were checked for quality and quantified using capillary electrophoresis. The libraries were sequenced on Illumina Hiseq 4000 sequencer as Single-Read 50 base reads following Illumina's instructions.