GSM6576609
Cells were pelleted by centrifugation and flash frozen with liquid nitrogen. Cell pellets were then shipped on dry ice. A human cell pellet from a blood sample was used as a positive control. Samples in 96-well plate were placed on dry ice and 3 times 150 ul of RNA shield (DNA/RNA Shield by Zymo, Cat. No. R1100-250) was added. The pellets were resuspended in RNA shield and the plate was transferred to room temperature and incubated at room temperature (RT) for 30 min. The plate was centrifuged for 4 min at 5,400 g. Supernatant was transferred to a new plate and remaining pellets were frozen. Qiagen's RNeasy Plus 96 Kit (cat. no. 74192) was used for RNA isolation. RLT buffer was supplemented with β-mercaptoethanol (β-ME) before use. 1 volume of RLT plus buffer was added to samples, and samples were mixed by vigorous shaking. The following steps were according to the RNeasy® Plus 96 Handbook, as available from https://www. qiagen.com/de/resources/download.aspx? id=4842d50e-a987-477a-a819-98a017445ccd& lang=en. RNA was eluted with 45 ul of RNAse free water. Next, samples were quantified with RiboGreen RNA Reagent, RediPlate™96 RiboGreen™RNA Quantitation Kit, cat. No. R11490. Libraries were prepared using an RNA Kit from Takara (Takara Bio USA, Inc. SMARTer® Stranded Total RNA- Seq Kit v2 - Pico Input Mammalian) according to the manufacturer's instructions. Libraries were then pooled and analyzed by Agilent 5200 Fragment Analyzer system (Agilent Technologies) for quantity and size distribution.