title: "Illumina sequencing of Human CD4 T cells RNA-Seq paired-end library  "
design:            Sequence capture probes were designed by Nimblegen (Madison, USA) to tile CCDS reference transcript for the following genes: BACH2, CAPZA1, CD69, CLEC16A, CTLA4, DEXI, FAM118A (included as a positive control known eQTL), ICOS, IFIH1, IL2, IL2RA, IL15RA, IL18RAP, IL21, KIAA1109, LSP1, MAPKAPK2, PFKFB3, PRKCQ, PTPN2, RBM17, RGS1, SH2B3, SOCS1, TAGAP. Probes were also designed to tile the intronic regions within each transcript, extending 40 bp into the exons on each side to capture retained intronic-exonic spanning reads since poly(A) selected RNA contains preRNA. Oligonucleotide probes (between 60 and 100 bp) were designed, excluding any sequences mapping to repeat regions or that were not unique in the human genome permitting 80% of the total 1.7 Mb genomic regions to be tiled. Probes were synthesised on 385,000 feature glass slide arrays.  Standard Solexa library preps were conducted according to the manufacturer’s instructions (Illumina, USA). For the RNA samples, a poly(A) selected RNA-seq library was constructed as described above. For the genomic DNA sample, DNA was extracted from whole blood using the Gentra PUREGENE DNA Extraction kit (QIAGEN, UK) according to manufacturer’s instructions. 5 µg of DNA was fragmented by sonication (Bioruptor, Diagenode, USA) before a standard paired-end library was constructed. After a single gel-extraction step to size select a 350 bp library, the adapter ligated library was amplified with 12 cycles of PCR. 1µg of either genomic DNA or RNA (cDNA) paired end Genome Analyzer library was added to 100 µg of Cot-1 DNA (Invitrogen, UK) with 1 µl each of the paired-end enhancing oligos (1 mM). The libraries were then hybridized to the sequence capture array for 72 hours at 42°C according to the manufacturer’s instructions. Arrays were stringently washed at 47.5ºC, and hybridized DNA eluted with NaOH. At multiple stages of the protocol, DNA size ranges were checked by Agilent Bioanalyzer and concentration by OD260/280 (Nanodrop, USA). Hybridization and elution of libraries was undertaken by Roche Nimblegen in Madison, USA.          