11 protocols
AccessionNameType
P-MTAB-26476
normalization data transformation protocol
Data was scanned according to manufacturer specifications.
P-MTAB-31816
nucleic acid library construction protocol
After 'chromatin_prep', 50 l 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' ends, the adapters were ligated to the ends of the DNA Fragments using 2 l of fourtyfold diluted Adapter oligo mix' in a total reaction volume of 25 l. 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 33.5 l of 10 mM tris buffer at pH7.0.
P-MTAB-26472
nucleic acid sequencing protocol
doi:10.1016/j.ymeth.2009.03.001; The PCR-product was sized fractionated on 2% agarose gel and a gel slice containing the 200-300 bp fragments was excised. The flowcells were prepared and processed according to the manufacturer' s protocols, with single-end sequencing for 36 to 45 cycles.
P-MTAB-26473
nucleic acid sequencing protocol
doi:10.1016/j.ymeth.2009.03.001; Subsequently, the DNA was amplified by 18 cycles of PCR, purified with QIAquick PCR purification Kit, and eluted with 33.5 l 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. The flowcells were prepared and processed according to the manufacturer' s protocols, with single-end sequencing for 36 to 45 cycles.
P-MTAB-31817
nucleic acid library construction protocol
Chromatin from an mass equivalent of 1/4 mouse liver was used for each ChIP experiment. 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' ends, the adapters were ligated to the ends of the DNA Fragments using 2 l of fourtyfold diluted Adapter oligo mix' in a total reaction volume of 25 l. Between these steps, the DNA was purified using the DNA Clean-and-Concentrator-5 kit (Zymo Research).
P-MTAB-26471
nucleic acid extraction protocol
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 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 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 l 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 C.
P-MTAB-26475
nucleic acid sequencing protocol
doi:10.1016/j.ymeth.2009.03.001; Subsequently, the DNA was amplified by 18 cycles of PCR, purified with QIAquick PCR purification Kit, and eluted with 33.5 l 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. The flowcells were prepared and processed according to the manufacturer' s protocols, with single-end sequencing for 36 to 45 cycles.
P-MTAB-31819
nucleic acid library construction protocol
After 'chromatin_prep', 50 l 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' ends, the adapters were ligated to the ends of the DNA Fragments using 2 l of fourtyfold diluted Adapter oligo mix' in a total reaction volume of 25 l. Between these steps, the DNA was purified using the DNA Clean-and-Concentrator-5 kit (Zymo Research).
P-MTAB-31818
nucleic acid library construction protocol
Chromatin from an mass equivalent of 1/4 mouse liver was used for each ChIP experiment. 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' ends, the adapters were ligated to the ends of the DNA Fragments using 2 l of fourtyfold diluted Adapter oligo mix' in a total reaction volume of 25 l. Between these steps, the DNA was purified using the DNA Clean-and-Concentrator-5 kit (Zymo Research).
P-MTAB-26474
nucleic acid sequencing protocol
doi:10.1016/j.ymeth.2009.03.001; Subsequently, the DNA was amplified by 18 cycles of PCR, purified with QIAquick PCR purification Kit, and eluted with 33.5 l 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. The flowcells were prepared and processed according to the manufacturer' s protocols, with single-end sequencing for 36 to 45 cycles.
P-MTAB-26470
growth protocol
doi:10.1016/j.ymeth.2009.03.001; Shortly, 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. Human material was obtained from Addenbrooke's Hospital, Cambridge licence (08/H0308/117) and the Liver Tissue Distribution Program at the University of Pittsburgh (NIDDK Contract # N01-DK-9-2310). Formaldehyde-crosslinked/flash frozen tissue was stored at -80 C at the CRI. Tc1 mice (annotated as Tc1_with_hschr21) were generated as described in (O'Doherty et al., 2005). Mice were initially obtained from Victor Tybulewic and Elizabeth Fisher and subsequently housed and bred within the Biological Resources Unit at CRI. Tc0 mice (annotated as Tc1_without_hschr21) are Tc1 sibling mice (ie the same genetic background) that have not inherited a copy of HsChr21.