GSM5202076: Sca1[+]/CD140a[-] cells Injury; Mus musculus; RNA-Seq
Total RNA was isolated from freshly sorted stromal cell populations using Trizol Reagent (Invitrogen 15596-026) according to manufacturer's recommendations. RNA quantitation was performed using Qubit™ RNA HS Assay Kit (Invitrogen Q32852). 20-100 ng of total RNA was employed to prepare mRNA library using QuantSeq 3'mRNA-Seq Library Prep Kit (Lexogen) according to manufacturer's standard protocol. Libraries were quantitated using Qubit dsDNA high sensitive quantitation kit (Invitrogen Q32854).  100 pM of library dilutions were prepared, barcoded and pooled using emulsion PCR using PI Hi-Q OT2 200 (Thermo Fisher Scientific) and Ion One Touch 2 (Thermo Fisher Scientific). After clonal DNA amplification, libraries were purified from magnetic beads using Ion One Touch ES device. Sequencing was accomplished using Ion PI Hi-Q Sequencing 200 reagents (Thermo Fisher Scientific A26434) on an Ion PI Chip v3 (Thermo Fisher A26771) and Ion Proton system (Thermo Fisher 4476610).