GSM3890942: OIR replicate 1; Mus musculus; RNA-Seq
Total RNA was extracted from cells stabilized in RNAprotect buffer 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 RNAprotect buffer at 2-8 °C. After pelleting, the RNAprotect buffer was replaced by RLT Plus and the samples were homogenized by vortexing for 30 sec. Genomic DNA contamination was removed using gDNA Eliminator spin columns. Next ethanol was added and the samples were applied to RNeasy MinElute spin columns followed by several wash steps. Finally 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 750 pg total-RNA. Double stranded cDNA was amplified by LD PCR (12 cycles) 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 fragmented) 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 SYBR FAST ABI Prism Library Quantification Kit (Kapa Biosystems, Inc., Woburn, MA, USA). Equimolar amounts of each library were pooled, and the pools were used for cluster generation on the cBot with the Illumina TruSeq SR Cluster Kit v3. The sequencing run was performed on an HiSeq 1000 instrument using the indexed, 50 cycles single-read (SR) protocol and the TruSeq SBS v3 Reagents according to the Illumina HiSeq 1000 System User Guide. Image analysis and base calling resulted in .bcl files, which were converted into .fastq.gz 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).
GEO Accession
GSM3890942