Instrument: Illumina HiSeq 2000
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: PAIRED
Construction protocol: Cells were harvested using TrypLE Express (Life Technologies) and medium removed by pelleting the cells (5 min at 1000 rpm). RNA was immediately stabilized by resuspending the cells in RNAprotect Cell Reagent (Qiagen) and RNaseOUT Recombinant Ribonuclease Inhibitor. (1:1000, Life Technologies). Prior to sorting, cells were diluted in PBS, pH 7.4 (no calcium, no magnesium, no phenol red, Life Technologies) and stained for viability using Hoechst 33342 (Life Technologies). 384-well capture plates were filled with 5µl of diluted Phusion HF buffer (1:500, New England Biolabs). Cells were sorted individually in each well of the 384-well capture plates using a FACSAria II flow cytometer (BD Biosciences) based on Hoechst DNA staining. After sorting, plates were immediately sealed, spin down and frozen on dry ice. Sorted cells were stored at -80°C. For lipid content based single cell sorting, cells were stained with HSC LipidTOX Neutral Lipid Stain (Life Technologies) and sorted according to their relatively “high” or “low” lipid content (upper/lower 20% or 50%). For bulk SCRB-Seq, populations of both unsorted and sorted cells were lysed in QIAzol (Qiagen) and RNA was extracted and purified using Direct-zol RNA MiniPrep (Zymo Research). Single Cell RNA Barcoding and Sequencing (SCRB-Seq), 3’ Digital Gene Expression (3’ DGE)