GSM8321134: WT Control 4; Mus musculus; RNA-Seq
cut a  small tissue of a frozen / fresh organ and transfer in to a 1,5 ml tube:  - under the hood add 500µl TROZOL , use a hand held homogenizer/potter until there are no tissue      chunks flying around      Info: blue Potter tip (single packaged) 2. drawer - Add 100µl chloroform, mix until cloudy by vortexing the sample for 15 sec (not less as 15sec!) Incubate sample for 3 min 28°C ( for cells sample 24°C) 300rpm 15min 12.000g 4°C  remove upper phase very carefully  into a 1,5ml Protein LB DNA/RNase free (Sarstedt Ref.: 72.706.000)         ca. 200µl supernatant - and throw away the rest (do not touch the middle phase)         keep chloroform for safety and dispose in special waste finally - Add 150µl isopropanol, mix samples gently by inversion several times Incubate for 10min 28°C ( for cells sample 24°C) 300rpm 10min 12.000g 4°C Decant supernatant Add 500µl 75% ETOH , and mix samples gently by inversion several times 5min 7500g 4°C Remove/ decant the supernatant completely , dry Cup wall by using paper, take care about the pellet      remove supernatant by using a pipette since it is loose. Dry Pellet 28°C for 10 – 15 min ( Pellet will become translucent from white). Resuspend pellet in Dnase, Rnase free water 25µl (10-50µl) – 25 ul than diluted to 125ul for Nanodrop Incubate for 10 min at 60°C Measure RNA concentration (Nanodrop) immediately or store at -80°C (directly -80°C without nitrogen) RNA sequencing (RNA-seq) libraries were performed using 200 ng total RNA of a non-stranded mRNA Seq (TruSeq RNA Library Preparation, #RS-122–2001)