GSM8945846: V4f_MG; Mus musculus; RNA-Seq
Cells were sorted into RNAprotect Cell Reagent (QIAGEN) according to the manufacturers recommendations. Total RNA was extracted from FACS sorted cells stabilized in RNAprotect Cell Reagent according to the “Purification of total RNA from animal and human cells” protocol of the RNeasy Plus Micro Kit (QIAGEN, Hilden, Germany). In brief, cells were stored and shipped in buffer RNAprotect at 2-8 °C. After pelleting by centrifugation for 5 minutes at 5,000 x g, the RNAprotect was replaced by 350 µl buffer RLT Plus and the samples were homogenized by vortexing for 30 seconds. Genomic DNA contamination was removed by using gDNA Eliminator spin columns. Next one volume of 70 % ethanol was added, and the samples were applied to RNeasy MinElute spin columns followed by several wash steps. Finally, the total RNA was eluted in 12 μl of nuclease free water. Purity and integrity of the RNA was assessed on the Agilent 2100 Bioanalyzer with the RNA 6000 Pico LabChip reagent set (Agilent, Palo Alto, CA, USA). Library preparation with Illumina Nextera XT Sample Preparation. The sequencing run was performed on a HiSeq 1000 instrument (Control Software v2.2.68) using the indexed, 50 cycles single-read (SR) protocol and the TruSeq SBS v3 Reagents according to the Illumina HiSeq 1000 System User Guide.