GSM9225937: S560s17_WT_laser_yes; Mus musculus; RNA-Seq
Total RNA was extracted from mouse choroid tissue stabilized in RNAlater buffer according to the “Purification of total RNA from animal and human tissue” protocol of the RNeasy Micro Kit (QIAGEN, Hilden, Germany). In brief, the tissue sample was stored in RNAlater and shipped at 2-8 °C. After centrifuging for 5 minutes at 5,000 x g, the RNAlater was removed and the sample was disrupted and homogenized in 350 µl RLT buffer containing 1% beta-mercaptoethanol with Precellys CK14 ceramic beads (1 cycle of 15 seconds at 5500 rpm) using a Precellys 24 Homogenisator (Bertin Corp., Rockville, MD, USA). Next the sample was centrifuged for 2 min at full speed and 350 µl of the cleared supernatant was transferred to a new tube. One volume of 70 % ethanol was added and the sample was applied to a RNeasy MinElute spin column followed by an on-column DNase digestion and several wash steps. Finally total RNA was eluted in 14 μl of nuclease free water. Purity and integrity of the RNA was assessed on the Agilent 2100 Bioanalyzer with the RNA 6000 Nano 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 10 ng of total-RNA was used to generate first strand cDNA. Double stranded cDNA was amplified by LD-PCR (8 cycles) and purified via magnetic bead clean-up. After validating the quality and quantity, approximately 500 pg of cDNA was enzymatically fragmented and stem-loop adapters were ligated. Then libraries were PCR amplified (14 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 one 100 cycles P3 Flow Cell with the dual index, paired-end (PE) run parameters. Image analysis and base calling were done by the Real Time Analysis Software (RTA) v3.10.30. The resulting .cbcl files were converted into .fastq files with the bcl2fastq v2.20 software.