title: GSM8483966: Liver, Rage-control, replicate 1; Mus musculus; RNA-Seq
library_construction_protocol: During FACS, the biliary epithelial cells sorted directly into RNA lysis buffer for subsequent RNA isolation. RNeasy Mini Kit was used to isolate the total RNA. Total RNA was subjected to an initial quality and quantity check using the BioAnalyzer by Agilent Technologies (RNA Pico Kit). Starting with 8.35 ng of input material total RNA was converted to cDNA using the SMART-Seq® v4 Ultra® Low Input RNA Kit for Sequencing (Takara Bio USA, Inc.) according to the manufacturer's instructions: During first-strand synthesis the product was primed by the 3' SMART-Seq CDS Primer II A and the SMART-Seq v4 Oligonucleotide was used for template switching at the 5' end of the transcript. The cDNA was then amplified by long distance PCR using 9 PCR cycles. The BioAnalyzer was used to check for successful cDNA synthesis and amplification (High Sensitivity DNA Kit). On the Covaris LE200 the amplified double-stranded cDNA was sheared to 150-250bp fragments. Successful fragmentation was verified on the TapeStation by Agilent Technologies (HS DNA 1000 Kit). For subsequent library preparation the NEBNext® ChIP-Seq Library Prep Master Mix Kit was used. After end repair, dA-Tailing and adaptor-ligation the cDNA was subjected to PCR amplification introducing i5 and i7 primers using 12 PCR cycles. Library quality was assessed on the TapeStation (DNA 1000 Kit). Sequencing was performed on the HiSeq 4000 platform (Illumina) in 100bp paired end mode, which resulted in at least 40 Mio reads/sample.