title: Age-dependent RGS5 loss in pericytes induces cardiac dysfunction and fibrosis in the heart (Bulk RNA-SEQ)
design: Age-dependent RGS5 loss in pericytes induces cardiac dysfunction and fibrosis in the heart (Bulk RNA-SEQ)
library_construction_protocol: Human Placenta Pericytes (hPC-PL, C-12980, PromoCell) were seeded at 6000 cells/cm2 density and culture in the respective Pericyte Growth Medium 2 (C-28041, PromoCell). RGS5 silencing was performed using siRNA. RGS5 siRNA (ID: SASI_Hs01_00094781, Sigma-Aldrich) and control siRNA (sense: CGUACGCGGAAUACUUCGA; antisense: UCGAAGUAUUCCGCGUACG) were used at a final concentration of 50nM. We used hPC-PL from different donors (464Z005, 463Z019.1, 422Z033.3). Total RNA was extracted from human placenta hPC-PL using the RNeasy Plus Mini Kit (Qiagen) according to the manufacturer's instructions. Total RNA was extracted from cells using the RNeasy Plus Mini Kit (Qiagen) according to the manufacturer's instructions. The synthesis of complementary DNA (cDNA) was performed by the reverse transcription (RT) of mRNA using M-MLV reverse transcriptase (Thermo-Fisher Scientific). Quantitative PCR reactions were performed using the StepOnePlus real-time PCR cycler (Thermo-Fisher Scientific) and relative gene expression was calculated by normalisation to ACTB gene expression (2-Ct). Human primers used: RGS5 (f: AGAAACCAGCCAAGACCCA, r: GGAGTTTGTCCAGGGAATCAC), ACTB (f: CATGTACGTTGCTATCCAGGC, r: CTCCTTAATGTCACGCACGAT), ACTA2 (f: CATCATGCGTCTGGATCTGG, r: GACAATCTCACGCTCAGCAG), TGFB2 (f: AAGAAGCGTGCTTTGGATGCGG, r: ATGCTCCAGCACAGAAGTTGGC). Total RNA was used as input for whole RNA-Seq library preparation, for which integrity was verified with LabChip Gx Touch 24 (CLS138162, Perkin Elmer). 1 µg of total RNA was used as input for Truseq Stranded total RNA library preparation following the low sample protocol (Illumina).