7e6e4184-875c-4152-9784-55fd7b685fcf
RNASeq of Ins-1E cells with internalised apoA-I (apoA-I positive) and Ins-1E cells without internalised apoA-I (apoA-I negative) from 3 independent experiments.
Ins-1E cells were incubated for 1 h at 37 ºC with unlabelled apoA-I (0.9 mg/mL) and Alexa Fluor 488-labelled apoA-I (0.1 mg/mL) under basal glucose conditions. The cells were harvested and incubated with anti-Alexa Fluor 488 IgG (4 µg/mL). They were then sorted based on the presence or absence of Alexa Fluor 488 fluorescence using a FACS Melody cell sorter (BD BioSciences, Franklin Lakes, NJ). Total RNA was collected and purified using RNeasy mini kit (Qiagen, Hilden, Germany). RNA was analysed for quality and integrity using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). Libraries were prepared from 500 ng of total RNA/sample using TruSeq Stranded mRNA-seq prep. Each library was single-read sequenced (1×75 bp) using NextSeq 500 High Output flowcell (60 million reads per sample).
Ins-1E cells were incubated for 1 h at 37 ºC with unlabelled apoA-I (0.9 mg/mL) and Alexa Fluor 488-labelled apoA-I (0.1 mg/mL) under basal glucose conditions. The cells were harvested and incubated with anti-Alexa Fluor 488 IgG (4 µg/mL). They were then sorted based on the presence or absence of Alexa Fluor 488 fluorescence using a FACS Melody cell sorter (BD BioSciences, Franklin Lakes, NJ). Total RNA was collected and purified using RNeasy mini kit (Qiagen, Hilden, Germany). RNA was analysed for quality and integrity using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). Libraries were prepared from 500 ng of total RNA/sample using TruSeq Stranded mRNA-seq prep. Each library was single-read sequenced (1×75 bp) using NextSeq 500 High Output flowcell (60 million reads per sample).