title: GSM8494495: Aorta_pericyte_spheroid_336; Homo sapiens; RNA-Seq
library_construction_protocol: Monolayer cells and spheroids formed on agarose were collected in Buffer RA1 (Takara Bio) +1% beta-mercaptoethanol (Fisher Scientific). NucleoSpin RNA kit from Takara Clontech was used for RNA extraction, 50ng input RNA used for library construction. RNA was assessed for quality using an TapeStation 4150 (Agilent) and RNA concentration was quantified on a Qubit FLEX fluorometer (Thermo Fisher). Libraries were generated from RNA samples with RIN scores >9 with the Illumina Stranded mRNA Library Prep kit (Illumina: 20040534) according to the manufacturer's instructions. Briefly, 50ng of input RNA was used for each sample. Following adapter ligation, 14 cycles of indexing PCR were completed, using IDT for Illumina RNA UD Indexes (Illumina: 20040553-6). Library quantification and assessment was conducted using a Qubit FLEX fluorometer and an Agilent TapeStation 4150. Libraries were normalized and pooled to 2 nM by calculating the concentration determined by fragment size (base pairs) and the concentration (ng/μL) of the libraries. Sequencing was performed on an Illumina NextSeq 2000, using a P2 100 flow cell. Sequencing was carried out on the pooled library (750 pM) with read lengths of 2x58 bp and a target of 40 million reads per sample. Sequencing data was demultiplexed by Illumina using the on-board DRAGEN FASTQ Generation software (v3.10.12). Library generation and sequencing was performed by the University of Pittsburgh Health Sciences Sequencing Core (HSSC), Rangos Research Center, UPMC Children's Hospital of Pittsburgh, Pittsburgh, Pennsylvania, United States of America.