title: Illumina HiSeq 2000 sequencing; Transcriptional profiling of acinar cells from mice engineered with 3 TFs for reprogramming to beta cells
design: Transcriptional profiling of acinar cells from mice engineered with 3 TFs for reprogramming to beta cells
library_construction_protocol: Pancreata were removed, perfused with 2 mg/mL collagenase P (Roche) in HBSS, minced and incubated in 37ďžĄC for 5 minutes. The cells were further dispersed by manual pipetting, washed with FACS staining buffer (R&D Systems), filtered through a 100 _m mesh cell strainer then centrifuged. Cell pellets were resuspended in AccuMax (Sigma) and DNase1 (3U, Ambion) and incubated at 37ďžĄC for 4 minutes. Afterwards, cells were washed with FACS staining buffer, centrifuged and resuspended in FACS buffer containing DNase1 (1 U/ml, Ambion) and 1mM EDTA. DAPI was used to stain for cell viability at a dilution of 1:1000.  Total RNA was isolated using TRIzol LS (Invitrogen) and column purification (Zymo Research), according to manufacturer's instructions. RNA was DNase-treated (Ambion) and integrity was assessed on the Bioanalyzer (Agilent).  Total RNA was amplified using the Ovation RNA-seq system (Nugen, 7100-08). The library was constructed using the Illumina TruSeq DNA sample prep kits A&B, FC-121-1001 and FC-121-1002 with the standard Illumina methods (Bentley 2008). Median library insert length=155; adapter length=100; amount amplified=10 ng.
attributes.tag: Experimental Factor: beta cell
attributes.value: cell type
attributes.tag: Experimental Factor: not applicable
attributes.value: days of DOX treatment