alias: mouseTA_experiment_0:0:0:0
title: Illumina Genome Analyzer IIx paired end sequencing; Erythroid cell nuclearRNA Rep1 experiment
design: Nuclear RNA characterization
library_construction_protocol: Genome-wide transcriptional output was characterized by sequencing a double-stranded cDNA library constructed from nuclear RNA (nucRNA). Following a hyper-osmotic swell in 0.1M sucrose, 0.1% Triton X-100 and 0.5 mM DTT, a single cell suspension was homogenised with a B-type Dounce. Intact nuclei were then separated from cytoplasmic debris through a 5 mM MgCl2, 10 mM Tris pH 8.0, 0.5 mM DTT, 0.33M sucrose cushion at 300g and re-suspended in 100 mM Tris-HCl pH 7.5, 100 mM NaCl, 30 mM MgCl2. RNA was purified from nuclear and cytoplasmic fractions using Trizol LS (Invitrogen) according to the suppliers instructions. Reverse transcription was performed using Superscript II (Invitrogen) and 10 ug random hexamer primers (Roche) per 500ng RNA. Second strand synthesis was performed using E. coli RNase H (Ambion) and E. coli DNA Polymerase I (NEB).  Library preparation was performed according to Illumina PE genomic protocol, incorporating improvements suggested in Quail et al 2008, with all reactions scaled according to starting DNA quantity
identifiers.namespace: BI-UK
identifiers.id: mouseTA_experiment_0:0:0:0
identifiers.namespace: The Babraham Institute, Cambridge, UK
identifiers.id: mouseTA_experiment_0:0:0:0