title: GSM5468508: classicMO_P9; Homo sapiens; RNA-Seq
library_construction_protocol: Total RNA was extracted from FACS sorted cells stabilized in RNAprotect buffer ac-cording 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 con-tamination 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). The SMARTer Ultra Low Input RNA Kit for Sequencing v4 (Clontech Laboratories, Inc., Mountain View, CA, USA) was used to generate first strand cDNA from approx-imately 1 ng total-RNA. Double stranded cDNA was amplified by LD PCR (12 cy-cles) and purified via magnetic bead clean-up. Library preparation was carried out as described in the Illumina Nextera XT Sample Preparation Guide (Illumina, Inc., San Diego, CA, USA). 150 pg of input cDNA were tagmented (tagged and frag-mented) by the Nextera XT transposome. The products were purified and amplified via a limited-cycle PCR program to generate multiplexed sequencing libraries. For the PCR step, 1:5 dilutions of index 1 (i7) and index 2 (i5) primers were used. The libraries were quantified using the KAPA Library Quantification Kit - Illumina/ABI Prism User Guide (Roche Sequencing Solutions, Inc., Pleasanton, CA, USA). Equimolar amounts of each library were sequenced on a NextSeq 500 instrument controlled by the NextSeq Control Software (NCS) v2.2.0, using two 75 Cycles High Output Kits with the dual index, single-read (SR) run parameters. Image analysis and base calling were done by the Real Time Analysis Software (RTA) v2.4.11. The resulting .bcl files were converted into .fastq files with the bcl2fastq v2.18 software. RNA extraction, library preparation and RNAseq were performed at the Genomics Core Facility “KFB - Center of Excellence for Fluorescent Bioanalytics” (University of Regensburg, Regensburg, Germany; www.kfb-regensburg.de).