Illumina Genome Analyzer IIx sequencing; IMR90_SeV
IMR90_SeV
IMR90 cells were cultured with MEM Earles media supplemented with 10% FBS 37C with 5% CO2 When cells are 80% confluent, Sendai Virus or media was added to the media and incubated for 6 hours at 37C. We have followed the protocol previously published in Core, L., Waterfall, J. & Lis, J. "Nascent RNA sequencing reveals widespread pausing and divergent initiation at human promoters." Science 322, 1845-1848 (2008). Briefly, nuclei from treated cells were isolated and incubated for 5 minutes with nucleotides and bromo-UTP analog. The nascent RNA from the labeling reaction was isolated and subjected to immunoaffinity purification using Br-dUTP antibody (Santa Cruz Biotech). After first round of purification of nascent RNA, the resulting RNA was ligated with 5' small RNA adaptor RNA (Illumina) and subjected to second Br-UTP purififcation. The resulting RNA is futher ligated using 3' adaptor RNA and subjected to third round of purification.  The final purified RNA was reverse transcribed and PCR amplified and size selected (150-250bp) using polyacrylamide gel electrophoresis to generate the final sequencing library. A modified small RNA library contruction protocol based on the Illumina kit was modified and incorporated with the EXTRACTION PROTOCOL. Please see the EXTRACTION PROTOCOL for details.
Experimental Factor: time
6 hour
Experimental Factor: infect
Sendai virus