title: Illumina HiSeq 4000 paired end sequencing; RNA-Seq profiling of ERCC1-XPF DNA repair defect (Er1F/-) versus wild type (Er1F/+) macrophages
design: RNA-Seq profiling of ERCC1-XPF DNA repair defect (Er1F/-) versus wild type (Er1F/+) macrophages
library_construction_protocol: Er1F/+ and Er1F/- BMDMs (bone marrow derived macrophages)were differentiated from bone marrow precursors. Briefly, bone marrow cells were isolated from mouse femurs and tibias and cultured for 7 days in DMEM containing 10% FBS, 30% L929 conditioned media, 50μg/ml streptomycin, 50 U/ml penicillin (Sigma) and 2mM L glutamine (Gibco). Total RNA was isolated from Er1F/+ and Er1F/- BMDMs (bone marrow derived macrophages) using a Total RNA isolation kit (Qiagen) as described by the manufacturer. Library preparation started with 1µg 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 to create the final cDNA libraries. After library validation and quantification (Agilent 2100 Bioanalyzer), equimolar amounts of all 12 libraries were pooled. The pool was quantified by using the Peqlab KAPA Library Quantification Kit and the Applied Biosystems 7900HT Sequence Detection System.
attributes.tag: Experimental Factor: genotype
attributes.value: wild type genotype