title: GSM4648602: Aorta_RNA Mouse, C66; Mus musculus; RNA-Seq
library_construction_protocol: After euthanasia, mouse ear and other tissues have been preserved in the TRIzol (Invitrogen) until further RNA isolation (n=4-5 per each experimental group). Arterial system and liver have been flushed with 1x cold PBS. Aorta has been isolated from the heart origin up to iliac bifurcation with perivascular fat removed along the whole distance. The RNA isolation was performed using the QIAGEN RNeasy kit according to manufacturer's protocol. The Starting volume was approx. 30mg and elution was 30ul. The library preparation was done using the QIAseq UPX 3' Transcriptome Kit (QIAGEN). A total of 10ng purified RNA was converted into cDNA NGS libraries. During reverse transcription, each cell is tagged with a unique ID and each RNA molecule is tagged with a unique molecular index (UMI). Then RNA was converted to cDNA. The cDNA was amplified, during the PCR indices were added and the libraries were purified. Library preparation QC was performed using Agilent Bioanalyzer. Based on quality of the inserts and the concentration measurements the libraries were pooled in equimolar ratios. The library pool(s) were quantified using qPCR. The library pool was then sequenced on a NextSeq500 sequencing instrument according to the manufacturer instructions with 100bp read length for read 1 and 27bp for read2. Raw data was de-multiplexed and FASTQ files for each pool was generated using the bcl2fastq software (Illumina inc.).