title: Illumina Genome Analyzer II sequencing; EnhanceBeta TCPA - Enhancer marks in sorted islet cells
design: EnhanceBeta TCPA - Enhancer marks in sorted islet cells
library_construction_protocol: Protocol after that of Tuteja et al. 2008 Nucleic Acid Research 36(12): 4149-4157. Islets (10,000 IEQs) were centrifuged (13,000Xg /30 sec) at room temperature (RT) and resuspended in 500ml 2.22% formaldehyde/PBS while rotating them for 10-min/RT. 59 ml 2.5M glycine was added to a final conc of 0.14 M to stop cross linking, followed by rotation for 5min/RT. Cells were centrifuged (13,000Xg/ 1 min) and pellet was washed with 1 ml PBS followed by centrifugation (13,000Xg) and then resuspended in 200 ml cold CHIP Whole cell lysis buffer. Cells were homogenized with small plastic pestle by hand and incubated on ice for 10 min. Cells were sonicated using Diagenode Bioruptor for 10 min on high at 30 s intervals, followed by centrifugation at 13,000Xg for 10 mins/40C. Sample was split and 10 ml of supernatant was used as input; remainder was frozen in liquid nitrogren. 2mg chromatin was added to 1mL CHIP dilution buffer and proteinase inhibitor. 2mg anti-histone antibody was then added followed by rotation at 4C/overnight. Protein G agarose beads were washed 3 times with 1 ml of CHIP dilution buffer, resuspended in BSA, protease inhibitor and CHIP dilution buffer for and rotated at 4C overnight. 100 ml of blocked agarose beads were added to each chromatin sample, incubate at 4C/1 hr followed by centrifugation at 13,000Xg to aspirate the supernatant. 1 ml of wash buffer was added (TSEI, TSEII, CHIP buffer 3 in consecutive order) to agarose pellet and rotate at RT/5min. 100ml elution buffer was added to the pellet and rotated for 15 min. This step was repeated and eluates were combined. 8 ml 5M NaCl/ 200 ml eluates were added and incubate at 65C overnight. 8 ml 1M Tris HCl (pH7.5), 0.5M EDTA and 1 ml 10mg/ml proteinase K, were added to the sample followed by incubation at 45 C /1hr for reverse cross-linking. Un-crosslinked chromatin was purified using PCR purification kit and eluted in 50 ml EB. Enrichment of CHIP samples was confirmed using qPCR using SYBR GreenER (Invitrogen) and Mx3000 PCR System (Stratagene, La Jolla, CA, USA). Enrichment of target region was calculated using control region (+60Kb of insulin gene) as a reference (lacking histone modifications) and by comparing input (sheared genomic DNA) to ChIP material. The immunoprecipitated DNA was modified for sequencing following the manufacturer protocol (Preparing Sample for ChIP Sequencing of DNA (11257047 Rev A)).
attributes.tag: Experimental Factor: cell type
attributes.value: beta cell
attributes.tag: Experimental Factor: immunoprecipitate
attributes.value: none