title: Illumina HiSeq 2500 paired end sequencing; RNA-seq of mouse DNA repair endonuclease XPF knock out versus wild type and versus trans retinoic acid (tRA) treated wild type
design: RNA-seq of mouse DNA repair endonuclease XPF knock out versus wild type and versus trans retinoic acid (tRA) treated wild type
library_construction_protocol: Primary  MEFs  were  isolated  from  E13.5  animals  and  cultured  in  standard  medium  containing  Dulbecco's  modified  Eagle medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS), 50μg/ml streptomycin, 50 U/ml penicillin (Sigma) and 2mM L glutamine (Gibco).  Cells were rinsed with PBS, exposed to tRA (10 μM) (Sigma-Aldrich) and cultured at 37oC for 16h.  Total RNA was isolated from cells using a Total RNA isolation kit (Qiagen) as described by the manufacturer. Libraries were prepared using the Illumina® TruSeq® mRNA stranded sample preparation kit. Library preparation started with 1ug total RNA. After poly-A selection (using poly-T oligo-attached magnetic beads), mRNA was purified and fragmented using divalent cations under elevated temperature. The RNA fragments underwent reverse transcription using random primers. This is followed by second strand cDNA synthesis with DNA Polymerase I and RNase H. After end repair and A-tailing, indexing adapters were ligated. The products were then purified and amplified (14 PCR cycles) to create the final cDNA libraries. After library validation and quantification (Agilent 2100 Bioanalyzer), equimolar amounts of library were pooled. The pool was quantified by using the Peqlab KAPA Library Quantification Kit and the Applied Biosystems 7900HT Sequence Detection System.