GSM8239894: Fibroblasts, Non-treated, rep5; Homo sapiens; RNA-Seq
Fibroblasts (F), myofibroblasts (M), and myofibroblasts treated with resting (R) or cytokine-primed (P) hMSC(AT)-CM were collected and preserved in RNAprotect Cell Reagent (Qiagen, Germany) and stored at − 80 °C until RNA isolation. RNA was isolated with the RNeasy Micro Kit (Qiagen, Germany) according to the manufacturer's protocol. RNA library preparation and sequencing were done by the molecular biology platform of the Institut de recherches cliniques de Montréal (IRCM). The RNA was quantified with a Qubit (Invitrogen), and RNA quality was measured on a BioAnalyzer chip (Agilent); the RNA integrity (RIN) was 7 or greater. Ribosomal RNA was depleted for 400 ng of total RNA (Ribocop depletion kit v 3.1; Lexogen), and libraries were prepared with the KAPA RNA hyperprep kit (Roche). Briefly, depleted RNA was reverse transcribed, the resulting cDNA was fragmented (94 °C 8 min), blunt-ended, and indexed adaptors were ligated. Ligated products were then amplified by PCR (9 cycles) and purified on KAPA pure beads (1:1 ratio). The final library had an average size distribution of 344 bp, and libraries were quantified by qPCR, and sequenced in paired-end 100 bp reads on Illumina NovaSeq 6000.