title: Illumina HiSeq 2000 paired end sequencing; Comparative transcriptomics of B cell transdifferentiation in human and mouse. RNA-Seq data
design: Comparative transcriptomics of B cell transdifferentiation in human and mouse. RNA-Seq data
library_construction_protocol: BLaER1 cells are as described here: https://doi.org/10.1016/j.celrep.2013.03.003 and are derived as a subclone of Seraphina cells CVCL_M646. C10 cells are as described https://doi.org/10.1016/j.stem.2009.10.004 and are derived as a subclone of the transformed fetal liver HAFTL-1 cell line. Fluorescent staining of cell surface markers was done with antibodies against Mac1 (APC) and CD19 (APC-Cy7) (BD Pharmigen). Samples were analyzed on the LSRII flow cytometer (BD Biosciences) and data were analyzed with FlowJo software (Tree Star). Human BLaER1 cells and mouse C10 cells were grown in RPMI (Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, and 100 U/ml penicillin G sodium. Transdifferentiation was induced in BlaER1 and C10 cells by addition of 100nM of β-estradiol (Calbiochem) and grown with 10nM of IL-3 and CSF-1 (Peprotech). Cells were lysed with Qiazol following the manufacturer's instructions (Qiagen). Chloroform was added to each sample and RNA contained in the aqueous fraction was isolated and purified by using RNeasy mini kit columns also following the manufacturer's instructions (Qiagen). Stranded poly A+ libraries were prepared with 1 ug of total RNA using TruSeq mRNA Sample Prep Kit (Illumina) according to manufacturer's protocol.