alias: E-MTAB-1294_update:input_HI_32_2
title: Transcriptional and epigenetic high throughput sequencing of human and mouse pancreatic islet-cells
design: Transcriptional and epigenetic high throughput sequencing of human and mouse pancreatic islet-cells
library_construction_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, around 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. 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)).
identifiers.namespace: Hospital Clinic de Barcelona, IDIBAPS, CIBERDEM
identifiers.id: E-MTAB-1294_update:input_HI_32_2