title: Human Islet HI32
organism: Homo sapiens
description: Protocols: Pancreatic Islets were isolated following the method of Bucher et al. (2005), Transplantation, 79: 91-97. Isolated islets were were cultured in CRML 1066 medium containing 10% FCS serum for shipping and, if not processed immediately, were recultured for 3 additional days. Cultured islets were rinsed with PBS buffer 3 times and cross-linked for 10 min in 1% formaldehyde at room temperature with constant shaking. After adding glycine (final concentration 125 mM), islets were rinsed with PBS-containing protease inhibitor cocktail (Roche) at 4 degreesC, snap frozen and stored at -80 degreesC. Islet purity was assessed by dithizone staining34 immediately before fixation. Protocol as described in Giresi et al. (2007), Genome Research, 17:877-855, with the following modifications: frozen pellets with ~3,000 cross-linked islets were thawed on ice in 1 ml lysis buffer (2% Triton X-100, 1% SDS buffer, 100 mM NaCl, 10 mM Tris-Cl at pH 8.0 and 1 mM EDTA buffer) and disrupted with five 1-min cycles using 0.5-mm glass beads (BioSpec). Samples were sonicated for 10-20 rounds of 30 pulses each (1 s on and 0.5 s off) using a Branson Sonifier 450D at 15% amplitude. After 10 rounds, the efficiency of sonication was assessed, and further rounds were performed when needed to ensure that the majority of chromatin fragments were in the 200-1,000-bp range. Debris was cleared by centrifugation at 15,000g for 5 min at 4 degreesC. Nucleosome-depleted DNA was extracted with phenol-chloroform followed by ethanol precipitation and RNase A (100 ?g/ml) treatment. Libraries were generated from gel-purified ~200-bp DNA fragments. Adapters were amplified using ligation-mediated PCR as detailed in Ren et al. (2000), Science, 290: 2306-2309.
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2014-12-23T17:01:35Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2018-03-08T22:39:58Z
attributes.tag: External Id
attributes.value: SAMEA3178837
attributes.tag: INSDC center name
attributes.value: Imperial Centre for Translational and Experimental Medicine
attributes.tag: INSDC first public
attributes.value: 2014-12-23T17:01:35Z
attributes.tag: INSDC last update
attributes.value: 2018-03-08T22:39:58Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-3199:Human Islet HI32
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell type
attributes.value: islet cell
attributes.tag: common name
attributes.value: human
attributes.tag: sample name
attributes.value: E-MTAB-3199:Human Islet HI32
attributes.tag: scientific_name
attributes.value: Homo sapiens