title: GSM8241424: Co-culture, Macrophage, 6 hours post myelin, mn:002; Mus musculus; RNA-Seq
library_construction_protocol: For RNA-seq samples, FACS-purified (non-) LLMs from MG and MDMs from primary PDG-Ink4a tumors, dTomato+ BMDMs from ex vivo assays (4.000-20.000 cells) or MES-like tumor cells from in vitro assays (20.000-100.000 cells) were homogenized in RLT buffer (79216, Qiagen), total RNA was isolatedusing the RNeasy Micro Kit (74004, Qiagen), including an on-column DNase digestion (79254, Qiagen),according to the manufacturer's instructions. Quality and quantity of the total RNA was assessed on the 2100 Bioanalyzer instrument following manufacturer's instructions “Agilent RNA 6000 Pico” (G2938-90046, Agilent Technologies).In general RNA yields of 3-135 ng total RNA and RNA integrity numbers (RIN) above 8 were obtained. RNA samples were concentrated with Agencourt RNAClean XP beads (A63987), according to manufacturer's instructions (Protocol 001298v001, Agencourt) with modifications. In short, 2.5-5 ng of total RNA for each sample was prepared in a volume of 30 μl by adding nuclease free water (AM9937, Ambion). 1.5 μl of Recombinant RNase inhibitor (2313A, Clontech) was added to each RNA sample. A 1.8x reaction volume RNAClean XP bead cleanup was performed, instead of adding 54 ul RNAClean XP bead suspension, 18 μl RNAClean XP bead suspension and 36 μl RNAClean XP bead buffer (same solution without the beads) were added to each RNA sample. After 3 times wash with 70% ethanol, the bead pellets were dried at 37 degrees Celsius for 3-5 minutes, untill bead pellet begins to show cracks. RNA was eluted by adding 4 μl of NF-H20 and mixing by vortexing. RNA library preparation was performed as previously described with modifications.96 In short, oligo dT primer hybridization was performed by the addition of Oligo dT mix (0.7ul H2O, 0.1 ul RNAse inhibitor (40U/ul), 0.1 ul dNTP mix (100mM) and 0.1 ul Oligo-dT30VN primer (100uM)) to the 4 ul sample-RNA bead suspension. Reverse transcription was performed as described but the MgCl2 concentration was adjusted to 10 mM. Template switching and 7-12 cycles pre-amplification of full length cDNAs with template switching oligo's was performed using ISPCR primer at a final concentration of 0.08 uM. The amplified full length cDNA was used for NGS library construction by Tagmentation for Illumina sequencing, using the Illumina Nextera XT DNA sample preparation kit (FC-131-1096, Illumina) and IDT® for Illumina Nextera DNA Unique Dual Indexes (20027216, Illumina). RNA sequencing libraries were quantified and normalized based library QC data generated on the Bioanalyzer system according to manufacturer's protocols (G2938-90321, Agilent Technologies).