7 protocols
Illumina HiSeq 2000
nucleic acid sequencing protocol
iCLIP protocol was followed to isolate RNA bound to CPSF-160 protein and amplify cDNA library as described in the manuscript. Specific barcodes were used for each RT reaction and are specified below for each sequencing run. Random barcodes were also incoperated into cDNAs to distinguish between PCR duplication of cDNA products. The final products were obtained by PCR with Illumina pair-end sequencing primers: 5-CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCT-3; 5-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3 Illumina HiSeq 2000
normalization data transformation protocol
Zhang, Y. et al. Model-based analysis of ChIP-Seq (MACS). Genome Biol 9, R137 (2008).
high throughput sequence alignment protocol
Fast alignment software
nucleic acid library construction protocol
The immunoprecipitated DNA was modified for sequencing following the Illumina sequencing following the manufacturer protocol (Preparing Sample for ChIP Sequencing of DNA (11257047 Rev A)).
nucleic acid extraction protocol
Human islets were fixed and sonicated as described previously (Nammo et al, 2012). Briefly, frozen crosslinked cell pellets of 4,000 islets were thawed on ice in 1 mL lysis buffer and disrupted with five 1-min cycles with 0.5 mm glass beads (BioSpec). Samples were sonicated for 10-20 cycles of 30 pulses each (1s on and 0.5 s off) using a Brandson Sonifier 450D at 15 % amplitude. The size for the bulk of chromatin obtained with this procedure was checked to be in the range from 200-1000 bp. For ChIPs, 300-400 human islet equivalents (IEQs) were pre-cleared with A/G sepharose beads (Ge Healthcare) for 1 h, rotating at 4 degrees C, and then incubated with each antibody. Afterwards, samples were rotated for 2 hr at 4 degrees C with protein A/G sepharose beads and then sequentially washed with low salt, high salt, LiCl and TE buffers. Next, chromatin was eluted with SDS 1% buffer and sequentially treated with RNAse (greater than 5 hr at 65 degrees C) and Proteinase K (overnight at 45 degrees C). Finally, DNA was extracted with phenol-chloroform followed by ethanol precipitation.
nucleic acid sequencing protocol
Illumina GAIIx. Sequencing Control Studio 2.8.