title: Illumina Genome Analyzer IIx paired end sequencing; Gene expression and alternative splicing analysis of breast carcinoma cells
design: Gene expression and alternative splicing analysis of breast carcinoma cells
library_construction_protocol: RNAs were purified from cultured cells using the High Pure RNA isolation kit (Roche). The integrity of RNA was checked with BioAnalyser 2100 (Agilent technologies) that indicated a RQI score >8 The libraries were prepared with Truseq mRNA Sample Prep kit (Illumina) from 1 microgram of total RNA following manufacturer's instructions. mRNAs were isolated by poly-A selection and fragmented (8 minutes at 94Â°). Fragmented mRNAs were used for reverse-transcription in the presence of Superscript II (Invitrogen) and random primers. After second strand synthesis, end-repair, A-tailing and purification, the double strand cDNA fragments were ligated to Truseq adapters containing the index sequences. Fifteen cycles of PCR in the presence of dedicated PCR primers and PCR master mix were applied to generate the final libraries.
attributes.tag: Experimental Factor: 1
attributes.value: replicate