GSM5852266: GAS_2; Mus musculus; RNA-Seq
To each GAS sample, 1 stainless steel bead together with 1ml of ice-cold TRIzol (15596018, Thermo Fisher Scientific, Rockford U.S.A.) was added. The mixtures were homogenized for 7 minutes using a bead mill at 50 rpm (TissueLyzer LT, 85600, Qiagen, Hilden Germany). The beads were removed using a forceps cleaned with RNAzap (AM9780/AM9782, Thermo Fisher Scientific, Rockford U.S.A.) after each transfer to avoid carryover of RNA. Addition of 200 μl of chloroform (22711.324, VWR international, Radnor U.S.A.) was followed by vigorous shaking for 15-20 sec and a centrifugation step of 15 min at 14,000 rcf at 4°C. The aqueous phase was transferred into a new vial and mixed with an equal amount of 100% ethanol (10821.365, VWR international, Radnor U.S.A.). RNA was then extracted using the RNA Clean & ConcentratorTM-25 Kit (R1017 & R1018, Zymo Research, Irvine USA). The whole volume was transferred to a Zymo-SpinTM IICR-column in a collection tube, spun down for 30 sec at 10000 rpm and the flow-through discarded. Next, a mixture of 5 μl of DNAse and 75 μl of DNA digestion buffer was added to the column and incubated for 15 min at room temperature. 400 μl of RNA Prep buffer was added directly on top of the DNase solution and the samples were spun down for 30 sec (4°C, 10,000 rpm). This process was repeated twice from the addition of 700 μl of RNA Wash buffer, spinning down for 30 sec at 4°C (10,000 rpm) and removal of flow through, followed by the addition of 400 μl of RNA wash buffer and a 2 min centrifugation step with the same settings. After the final removal of flow through, 30 μl of DNase/RNase-Free Water was added directly onto the colum matrix. The colum was placed in a fresh vial and once again spun down for 30 seconds (4°C, 10000 rpm). The RNA quality was measured with a Fragment Analyzer (Agilent, Santa Clara, California, USA) and only those samples with a 28S/18S ratio over 1.9 were used for library construction which was outsourced to Novogene. Total mRNA was sequenced PE150 on a Novaseq.