GSM9487419: M2_Veh_Stroke_tdT-; 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 and RNAseq were carried out as described in the SMART-Seq mRNA LP User Manual (Takara Bio, Inc., CA, USA), the Illumina NextSeq 2000 Sequencing System Guide (Illumina, Inc., San Diego, CA, USA), and the KAPA Library Quantification Kit - Illumina/ABI Prism Protocol (Roche Sequencing Solutions, Inc., Pleasanton, CA, USA). In brief, approximately 500 pg of total-RNA was used to generate first strand cDNA. Double stranded cDNA was amplified by LD-PCR (13 cycles) and purified via magnetic bead clean-up. After validating the quality and quantity, 250 pg of cDNA was enzymatically fragmented and stem-loop adapters were ligated. Then libraries were PCR amplified (16 cycles) and indexed, generating Illumina-compatible libraries with unique dual indexes (UDIs). After a magnetic bead purification, the libraries were quantified using the KAPA Library Quantification Kit. Equimolar amounts of each library were sequenced on an Illumina NextSeq 2000 instrument controlled by the NextSeq 2000 Control Software (NCS) v1. 5.0.42699, using two 100 cycles P2 Flow Cells with the dual index, single-read (SR) run parameters.