|Public on May 28, 2015
|cell type: Jurkat
|Human Jurkat cells were purchased from the American Type Culture Collection (ATCC), Manassas, VA. Cells were maintained under typical conditions in RPMI media with 10% Bovine Calf Serum. For location analysis, cells were grown to a density of 1 million per ml and 99% viability prior to cross-linked with formaldehyde for 10 min.
|Using 10 μg of total RNA, we prepared sequencing libraries according to the following protocol. Polyadenylated RNA was purified by two rounds of selection with Dynabeads mRNA Purification Kit for mRNA Purification from total RNA (Life Technologies, 610-06) following the manufacturer instructions. This resulting RNA was then further processed for RNA-Seq assays. Briefly, polyadenylated RNA was fragmented with divalent cations under elevated temperature. First strand cDNA synthesis was performed with random hexamers and Superscript III reverse transcriptase (Life Technologies, 18080-051). Second strand cDNA synthesis was performed by using RNase H and DNA Polymerase I. In the second-strand synthesis reaction, dTTP was replaced with dUTP. After cDNA synthesis, the double-stranded products were end repaired, a single “A” base was added, and Illumina PE adaptors were ligated onto the cDNA products. The ligation products with an average size of 300 bp were purified by using agarose gel electrophoresis. Following gel purification, the strand of cDNA containing dUTP was selectively destroyed during incubation of purified double-stranded DNA with HK-UNG (Epicenter, HU59100). The adaptor ligated single-stranded cDNA was then amplified with 15 cycles of PCR and PCR products were purified by using gel electrophoresis. These RNA-Seq libraries were subsequently sequenced on Illumina HiSeq 2000. Sequences were aligned by using Bowtie (version 0.12.2) to build version HG19 of the human. The RPKM (reads per kilobase of exon per million) was then computed for each gene.
Total RNA was purified using mirVana miRNA isolation kit (Life technologies) following the manufacturer instructions.
|Illumina HiSeq 2000
|Aligned using TopHat v2.0.13 with configuration: library-type fr-firststrand -p 40 --microexon-search --coverage-search
Supplementary_files_format_and_content: Aligned reads were first separated into strand/direction using samtools view. Density of strand-separated reads in 50bp bins was calculated using genomeCoverageBed. The union of the resulting bedGraph files was taken using unionBedGraphs and converted to WIG format using bedgraph_to_wig.pl (https://github.com/svigneau/)
|May 18, 2015
|Last update date
|May 15, 2019
|Richard A Young
|Whitehead Institute for Biomedical Research
|9 Cambridge Center
|Activation of proto-oncogenes by disruption of chromosome neighborhoods [RNA-Seq]
|Activation of proto-oncogenes by disruption of chromosome neighborhoods