title: GSM4763865: Stim_fibroblasts; Mus musculus; RNA-Seq
library_construction_protocol: Murine lungs were dissociated and the entire cell suspension was then plated on appropriately sized tissue culture plasticware. Fibroblasts were cultured in advanced DMEM/F12 (#12634010, Thermo Fisher Scientific), with 10 % vol/vol FBS (HyClone) or to induce a lipofibroblast like phenotype with the addition of 1 % vol/vol ITS+3 liquid media supplement (#I2771, Merck Milipore, Burlington, MA, USA) to the culture media. Media was changed every other day and the cells sub cultured when 80 % confluent. Fibroblasts cultured to passage three (P3) were considered free of contaminating cells based on previous experience in the laboratory. At P3 the cultured fibroblasts were then stimulated with one or a combination (as indicated) of 10 μM rosiglitazone (#72622, Stem Cell Technologies, Cambridge, MA, USA), 1 μM SB4315442 (#1614, R&D Systems, Minneapolis, MO, USA), 4 μM, rhBMP4 (#314-BP, R&D Systems) for 14 days. Matched control cells isolated from the same lung were stimulated in an identical media, for an identical duration, with the appropriate vehicle. Library preparation and sequencing were performed as our previously published papers. Sequencing libraries for cDNA from single cells were prepared as per the Single Cell 3′ v2 Reagent Kits User Guide (10x Genomics, Pleasanton, CA, USA). Cellular suspensions were loaded on a Chromium Controller instrument (10x Genomics) to generate single-cell Gel Bead-In-EMulsions (GEMs). GEM-reverse transcription (RT) was performed in a Veriti 96-well thermal cycler (Thermo Fisher Scientific, Waltham, MA, USA). GEMs were collected and the cDNA was amplified and purified with SPRIselect Reagent Kit (Beckman Coulter, Brea, CA, USA). Indexed sequencing libraries were constructed using Chromium Single-Cell 3′ Library Kit for enzymatic fragmentation, end-repair, A-tailing, adapter ligation, ligation cleanup, sample index PCR, and PCR cleanup. The barcoded sequencing libraries were quantified by quantitative PCR using the KAPA Library Quantification Kit for Illumina platforms (KAPA Biosystems, Roche Holding AG, Basel, Switzerland). Sequencing libraries were loaded on a NextSeq500 (Illumina, San Diego, CA, USA) with a custom sequencing setting (26bp for Read 1 and 98bp for Read 2) to obtain a sequencing depth of ~200K reads per cell.
attributes.tag: GEO Accession
attributes.value: GSM4763865