hsa1308
Homo sapiens
Protocols: doi:10.1016/j.ymeth.2009.03.001; Liver material was crosslinked in 1% formaldehyde for 20 minutes followed by a 10 minute incubation in 500 mM glycine buffer to neutralize the formaldehyde. Samples were rinsed with PBS and frozen until use. doi:10.1016/j.ymeth.2009.03.001; Crosslinked animal livers were dounce homogenized into a single cell suspension and rinsed with ice cold PBS. Each pellet of cross-linked material was resuspended in 10 ml of LB1 (50 mM Hepes-KOH, pH 7.5; 140 mM NaCl; 1mM EDTA; 10% Glycerol; 0.5% NP-40 or Igepal CA-630; 0.25% Triton X-100) and pelleted again by centrifugation at 2,000 x rcf for 4 minutes at 4 degrees C. Pellets were then rinsed in 10 ml of LB2 (10 mM Tris-HCL, pH8.0; 200 mM NaCl; 1 mM EDTA; 0.5 mM EGTA) and pelleted again by centrifugation at 2,000 x rcf for 4 minutes at 4 degrees C. The nuclei preparation was resuspended in 3 ml LB3 (10 mM Tris-HCl, pH 8; 100 mM NaCl; 1 mM EDTA; 0.5 mM EGTA; 0.1% Na-Deoxycholate; 0.5% N-lauroylsarcosine). Chromatin in the nuclei fraction was fragmented to an average length of 300 bp by sonication. After sonication 300 microlitreof 10% Triton X-100 was added to the 3 ml of sonicated lysate and debris was removed by centrifugation at 20,000 x rcf for 10 minutes at 4 degrees C. doi:10.1016/j.ymeth.2009.03.001; Crosslinked livers were dounce homogenized into a single cell suspension and rinsed with ice cold PBS. Each pellet of cross-linked material was resuspended in 10 ml of LB1 (50 mM Hepes-KOH, pH 7.5; 140 mM NaCl; 1mM EDTA; 10% Glycerol; 0.5% NP-40 or Igepal CA-630; 0.25% Triton X-100) and pelleted again by centrifugation at 2,000 x rcf for 4 minutes at 4 degrees Celcius. Pellets were then rinsed in 10 ml of LB2 (10 mM Tris-HCL, pH8.0; 200 mM NaCl; 1 mM EDTA; 0.5 mM EGTA) and pelleted again by centrifugation at 2,000 x rcf for 4 minutes at 4 degrees Celcius. The nuclei preparation was resuspended in 3 ml LB3 (10 mM Tris-HCl, pH 8; 100 mM NaCl; 1 mM EDTA; 0.5 mM EGTA; 0.1% Na-Deoxycholate; 0.5% N-lauroylsarcosine). Chromatin in the nuclei fraction was fragmented to an average length of 300 bp by sonication. After sonication 300ul of 10% Triton X-100 was added to the 3 ml of sonicated lysate and debris was removed by centrifugation at 20,000 x rcf for 10 minutes at 4 degrees Celcius. After 'chromatin_prep', 50ul of cell lysate from each sonication was removed as the input control DNA that was then processed as outlined in the 'chip_seq' protocol. Solexa libraries were prepared following the instructions of Illumina (Sample preparation for genomic DNA version 2.2) with the following modifications. The ChIP-enriched DNA and input DNA were not further fragmented. After end-repair and addition of an A base to the 3-prime ends, the adapters were ligated to the ends of the DNA Fragments using 2ul of fourtyfold diluted Adapter oligo mix in a total reaction volume of 25ul. Between these steps, the DNA was purified using the DNA Clean-and-Concentrator-5 kit (Zymo Research). Subsequently, the DNA was amplified by 18 cycles of PCR, purified with QIAquick PCR purification Kit, and eluted with 33.5ul of 10 mM tris buffer at pH7.0. The PCR-product was sized fractionated on 2% agarose gel and a gel slice containing the 200-300 bp fragments was excised.
ENA-FIRST-PUBLIC
2014-09-27T17:03:45Z
ENA-LAST-UPDATE
2018-03-08T16:18:28Z
External Id
SAMEA2054749
INSDC center name
Cambridge Research Institute
INSDC first public
2014-09-27T17:03:45Z
INSDC last update
2018-03-08T16:18:28Z
INSDC status
public
Submitter Id
E-MTAB-1509:hsa1308
broker name
ArrayExpress
common name
human
developmental stage
adult
individual
hsa1308
organism part
liver
sample name
E-MTAB-1509:hsa1308
scientific_name
Homo sapiens
sex
male