Illumina HiSeq 2000 paired end sequencing; Transcriptomic profiling of human BLaER1 pre-B cells transdifferentiation into macrophages
Transcriptomic profiling of human BLaER1 pre-B cells transdifferentiation into macrophages
Cells were collected at twelve time points after transdifferentiation induction: 0, 3, 6, 9, 12, 18, 24, 36, 48, 72, 120 and 168 hours. Cell pellets were frozen prior to RNA extraction. Human BLaER1 cells were grown in RPMI medium (Invitrogen) supplemented with 10% heat-inactivated foetal bovine serum (FBS, Gibco), 2 mM L-glutamine, and 100 U/ml penicillin G sodium (Gibco). Cells were maintained at 37ºC in a humid atmosphere containing 5% CO2. Transdifferentiation was induced by addition of 100 nM of beta-estradiol (Calbiochem) and 10 nM of IL-3 and CSF-1 (Peprotech). Cells were lysed with QiAzol (Qiagen, The Netherlands). Chloroform was added to each sample, and RNA contained in the aqueous solution was isolated and purified by using RNeasy mini kit columns (Qiagen, The Netherlands). Poly A+ libraries were prepared with 1 ug of total RNA and using TruSeq Stranded mRNA Library Prep Kit (Illumina, USA) according to the manufacturer's protocol.