PMC:4157143 / 5786-7208 JSONTXT

{"project":"2_test","denotations":[{"id":"25192044-19620212-2055624","span":{"begin":871,"end":873},"obj":"19620212"}],"text":"Cell Culture and RNA Sequencing\nEpstein-Barr-virus-transformed peripheral blood B lymphocytes (catalog no. XC01463) from families from the CEU population (Utah residents with ancestry from northern and western Europe from the CEPH collection) were purchased from the Coriell Institute and grown in RPMI 1640 supplemented with 10% fetal calf serum and penicillin and streptomycin in humidified 5% CO2 at a concentration of ∼1 × 106 cells/ml. Total RNA was isolated with Trizol. RNA quality was assessed with the Agilent Bioanalyzer 2100, and RNA integrity numbers above 9 were used for cDNA production. One microgram of total RNA was used for isolating polyA-purified mRNA and subsequently used for cDNA-library construction with the Illumina TruSeq RNA Preparation Kit. Strand specificity was performed with 2'-deoxyuridine 5'-triphosphate during second-strand synthesis.27 All samples were indexed with Illumina adapters and sequenced with an Illumina HiScanSQ. We subsequently sequenced each cDNA library on an Illumina HiSeq to obtain 30 million 75 bp paired-end reads per individual. We performed RNA sequencing (RNA-seq) for all 17 individuals (all three generations); however, for eQTL association, we only used the 11 children, and for ASE analysis, we used the two parents and 11 children. All RNA-seq data for all 17 individuals are freely available at the Gene Expression Omnibus under accession number GSE56961."}